Non-invasive localization of a light-emitting conjugate in a mammal

ABSTRACT

Methods and compositions for detecting and localizing light originating from a mammal are disclosed. Also disclosed are methods for targeting light emission to selected regions, as well as for tracking entities within the mammal. In addition, animal models for disease states are disclosed, as are methods for localizing and tracking the progression of disease or a pathogen within the animal, and for screening putative therapeutic compounds effective to inhibit the disease or pathogen.

This application is a divisional application of U.S. Ser. No.09/233,698, filed 19 Jan. 1999, now U.S. Pat. No. 6,649,143, which is acontinuation of U.S. Ser. No. 08/602,396, filed 16 Feb. 1996, nowabandoned, which is a continuation-in-part of U.S. Ser. No. 08/270,631,filed 1 Jul. 1994, now U.S. Pat. No. 5,650,135, from which priority isclaimed under 35 U.S.C. § 120, and which applications are hereinincorporated by reference in their entireties. This application isrelated to U.S. Ser. No. 09/233,507, filed 19 Jan. 1999, now U.S. Pat.No. 6,217,847, herein incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This work was supported in part by grants from the Office of NavalResearch (N-00014-91-C-0170), the National Institutes of Health (PHSM01-RR-00070-30/1 and RR-00081) and the United States Public HealthService. Accordingly, the United States Government has certain rights inthis invention.

I. FIELD OF THE INVENTION

The present invention relates to noninvasive methods and compositionsfor detecting, localizing and tracking light-emitting entities andbiological events in a mammalian subject.

II. BACKGROUND OF THE INVENTION

The ability to monitor the progression of infectious diseases is limitedby the current ex vivo methods of detecting and quantifying infectiousagents in tissues. The replication of an infectious agent in a hostoften involves primary, secondary and tertiary sites of replication. Thesites of replication and the course that an infectious agent followsthrough these sites is determined by the route of inoculation, factorsencoded by the host as well as determinants of the infecting agent.

Experience may offer, in some cases, an estimate of probable sites ofreplication and the progress of an infection. It is more often the case,however, that the sites of infection, and the pace of the disease areeither not known or can only roughly be estimated. Moreover, theprogression of an infectious disease, even in inbred strains of mice, isoften individualized, and serial, ex vivo analyses of many infectedanimals need to be conducted to determine, on the average, what course adisease will follow in an experimentally infected host.

Accordingly, it would be desirable to have a means of tracking theprogression of infection in an animal model. Ideally, the tracking couldbe done non-invasively, such that a single animal could be evaluated asoften as necessary without detrimental effects. Methods and compositionsof the present invention provide a non-invasive approach to detect,localize and track a pathogen, as well as other entities, in a livinghost, such as a mammal.

III. SUMMARY OF THE INVENTION

In one embodiment, the invention includes a noninvasive method fordetecting the localization of a biocompatible entity in a mammaliansubject. The entity can be a molecule, macromolecule, cell,microorganism (including a pathogen), a particle, or the like.

The method includes administering to the subject a conjugate of theentity and a light-generating moiety. Light-generating moieties aretypically molecules or macromolecules that give off light. They maygenerate light as a result of radiation absorption (e.g., fluorescent orphosphorescent molecules), or as a result of a chemical reaction (e.g.,bioluminescent proteins). Exemplary light-generating moieties arebioluminescent proteins, such as luciferase and aequorin, and colored orfluorescent proteins, such as yellow fluorescent protein and ferredoxinIV.

The moiety may be conjugated to the entity by a variety of techniques,including incorporation during synthesis of the entity (e.g., chemicalor genetic, such a fusion protein of an antibody fragment and alight-generating protein), chemical coupling post-synthesis,non-covalent association (e.g., encapsulation by liposomes), in-situsynthesis in the entity (e.g., expression of a heterologousbioluminescent protein in a transformed cell), or in situ activatablepromoter-controlled expression of a bioluminescent protein in cells of atransgenic animal stimulated by a promoter inducer (e.g.,interferon-activated promoter stimulated by infection with a virus).

After a period of time in which the conjugate can localize in thesubject, the subject is immobilized within the detection field of aphotodetector device for a period of time effective to measure asufficient amount of photon emission (with the photodetector device) toconstruct an image. An exemplary photodetector device is an intensifiedcharge-coupled device (ICCD) camera coupled to an image processor. Ifthe image can be constructed in a time short relative to the time scaleat which an “unimmobilized” subject moves, the subject is inherently“immobilized” during imaging and no special immobilization precautionsare required. An image from the photon emission data is thenconstructed.

The method described above can be used to track the localization of theentity in the subject over time, by repeating the imaging steps atselected intervals and constructing images corresponding to each ofthose intervals.

The method described above can be used in a number of specificapplications, by attaching, conjugating or incorporating targetingmoieties onto the entity. The targeting moiety may be an inherentproperty of the entity (e.g., antibody or antibody fragment), or it maybe conjugated to, attached to, or incorporated in the entity (e.g.,liposomes containing antibodies). Examples of targeting moieties includeantibodies, antibody fragments, enzyme inhibitors, receptor-bindingmolecules, various toxins and the like. Targets of the targeting moietymay include sites of inflammation, infection, thrombotic plaques andtumor cells. Markers distinguishing these targets, suitable forrecognition by targeting moieties, are well known.

Further, the method may be used to detect and localize sites ofinfection by a pathogen in an animal model, using the pathogen (e.g.,Salmonella) conjugated to a light-generating moiety as the entity.

In a related embodiment, the invention includes a noninvasive method fordetecting the level of a biocompatible entity in a mammalian subjectover time. The method is similar to methods described above, but isdesigned to detect changes in the level of the entity in the subjectover time, without necessarily localizing the entity in the form of animage. This method is particularly useful for monitoring the effects ofa therapeutic substance, such an antibiotic, on the levels of an entity,such as a light-emitting bacterium, over time.

In another embodiment, the invention includes a noninvasive method fordetecting the integration of a transgene in a mammalian subject. Themethod includes administering to the subject, a vector constructeffective to integrate a transgene into mammalian cells. Such constructsare well known in the art. In addition to the elements necessary tointegrate effectively, the construct contains a transgene (e.g., atherapeutic gene), and a gene encoding a light-generating protein underthe control of a selected activatable promoter. After a period of timein which the construct can achieve integration, the promoter isactivated. For example, if an interferon inducible promoter is used, apoly-inosine and -cytosine duplex (poly-IC) can be locally administered(e.g., footpad injection) to stimulate interferon production. The HIVLTR could similarly be used and induced, for example, withdimethylsulfoxide (DMSO). The subject is then placed within thedetection field of a photodetector device, such as an individual wearinglight-intensifying “night vision” goggles, and the level of photonemission is measured, or evaluated. If the level is above background(i.e., if light can be preferentially detected in the “activated”region), the subject is scored as having integrated the transgene.

In a related embodiment, the invention includes a noninvasive method fordetecting the localization of a promoter-induction event in an animalmade transgenic or chimeric for a construct including a gene encoding alight-generating protein under the control of an inducible promoter.Promoter induction events include the administration of a substancewhich directly activates the promoter, the administration of a substancewhich stimulates production of an endogenous promoter activator (e.g.,stimulation of interferon production by RNA virus infection), theimposition of conditions resulting in the production of an endogenouspromoter activator (e.g., heat shock or stress), and the like. The eventis triggered, and the animal is imaged as described above.

In yet another embodiment, the invention includes pathogens, such asSalmonella, transformed with a gene expressing a light-generatingprotein, such as luciferase.

In another aspect, the invention includes a method of identifyingtherapeutic compounds effective to inhibit spread of infection by apathogen. The method includes administering a conjugate of the pathogenand a light-generating moiety to control and experimental animals,treating the experimental animals with a putative therapeutic compound,localizing the light-emitting pathogen in both control and experimentalanimals by the methods described above, and identifying the compound astherapeutic if the compound is effective to significantly inhibit thespread or replication of the pathogen in the experimental animalsrelative to control animals. The conjugates include afluorescently-labeled antibodies, fluorescently-labeled particles,fluorescently-labeled small molecules, and the like.

In still another aspect, the invention includes a method of localizingentities conjugated to light-generating moieties through media ofvarying opacity. The method includes the use of photodetector device todetect photons transmitted through the medium, integrate the photonsover time, and generate an image based on the integrated signal.

In yet another embodiment, the invention includes a method of measuringthe concentration of selected substances, such as dissolved oxygen orcalcium, at specific sites in an organism. The method includes entities,such as cells, containing a concentration sensor—a light-generatingmolecule whose ability to generate light is dependent on theconcentration of the selected substance. The entity containing thelight-generating molecule is administered such that it adopts asubstantially uniform distribution in the animal or in a specific tissueor organ system (e.g., spleen). The organism is imaged, and theintensity and localization of light emission is correlated to theconcentration and location of the selected substance. Alternatively, theentity contains a second marker, such as a molecule capable ofgenerating light at a wavelength other than the concentration sensor.The second marker is used to normalize for any non-uniformities in thedistribution of the entity in the host, and thus permit a more accuratedetermination of the concentration of the selected substance.

In another aspect, the invention includes a method of identifyingtherapeutic compounds effective to inhibit the growth and/or themetastatic spread of a tumor. The method includes (i) administeringtumor cells labeled with or containing light-generating moieties togroups of experimental and control animals, (ii) treating theexperimental group with a selected compound, (iii) localizing the tumorcells in animals from both groups by imaging photon emission from thelight-generating molecules associated with the tumor cells with aphotodetector device, and (iv) identifying a compound as therapeutic ifthe compound is able to significantly inhibit the growth and/ormetastatic spread of the tumor in the experimental group relative to thecontrol group.

These and other objects and features of the invention will be more fullyappreciated when the following detailed description of the invention isread in conjunction with the accompanying drawings.

IV. BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B and 1C show a map of the lux pCGLS1 plasmid used totransform Salmonella strains SL1344, BJ66 and LB5000 to generate strainsSL1344lux, BJ66lux and LB5000lux. FIG. 1A depicts a restriction enzymemap of the lux operon, which is inserted into the BamHI site of thepolylinker depicted in FIG. 1B. A sequence included in the multiplecloning site (MCS) is provided in FIG. 1B, with the Bam HI siteindicated in bold type. A graphical representation of a pUC18 vectorwithout insert is shown in FIG. 1C.

FIGS. 2A, 2B, 2C, 2D and 2E depict the adherence and invasion ofSalmonella strains SL1344lux and BJ66lux on macrophages and HEp-2 cells.

FIG. 2A depicts luminescent bacterial cells localized in wells of anassay dish. The pseudo-color image, obtained by integrating photons overone minute, is superimposed over a gray scale image of the assay dish,producing the “composite image” shown.

FIG. 2B depicts the relative light intensity of wells that were nottreated with gentamicin.

FIG. 2C depicts the number of colony forming units (CFU) per ml isolatedfrom the same wells as were imaged in FIG. 2B.

FIG. 2D depicts the relative light intensity of wells that were treatedwith gentamicin.

FIG. 2E depicts the number of colony forming units (CFU) per ml isolatedfrom the same wells as were imaged in FIG. 2D.

FIG. 3A depicts a composite image of four glass capillary tubescontaining dilutions of LB5000lux bacterial suspensions. Luminescencewas determined by integrating over 30 seconds. Air pockets are presentin each tube on both sides of the suspension.

FIG. 3B depicts the distribution of bioluminescence followingintraperitoneal inoculation of wild-type SL1344lux into mice.

FIG. 4 depicts the effect of human blood on the light emission frombioluminescent Salmonella.

FIG. 5 depicts a schematic diagram of a vial used to test thetransmission of light generated by LB5000lux through animal tissue.

FIG. 6A depicts composite images of Balb/c mice orally inoculated withlow virulence LB5000lux Salmonella , and imaged at the times indicated.The luminescence signal was integrated over 5 minutes.

FIG. 6B depicts composite images of Balb/c mice orally inoculatedwithnon-invasive BJ66lux Salmonella , and imaged at the times indicated.The luminescence signal was integrated over 5 minutes.

FIG. 6C depicts composite images of Balb/c mice orally inoculated withvirulent SL1344lux Salmonella , and imaged at the times indicated. Theluminescence signal was integrated over 5 minutes.

FIG. 7 depicts a composite image showing the distribution of Salmonellain mice 32 hours following intraperitoneal (i.p.) injections with eithervirulent SL1344lux (left two animals) or low virulence LB5000lux (righttwo animals) strains of the bacterium.

FIG. 8A depicts the distribution of virulent Salmonella in miceresistant to systemic Salmonella infections (129×Balb/c, Ity^(r/s)) onday one (1).

FIG. 8B depicts the distribution of virulent Salmonella in miceresistant to systemic Salmonella infections (129×Balb/c, Ity^(r/s)) onday eight (8).

FIGS. 9A, 9B, and 9C depict the distribution of mutant Salmonella withreduced virulence (BJ66lux) seven days following oral inoculation.

FIG. 9A depicts external, non-invasive imaging of the luminescence.

FIG. 9B depicts the same animal imaged following laparotomy. Labeledorgans are cecum (C), liver (L), small intestine (I), and spleen (Sp).

FIG. 9C depicts a post-laparotomy image generated following injection ofair into the lumen of the intestine both anterior and posterior to thececum.

FIGS. 10A, 10B and 10C depict the distribution of Salmonella SL1344luxin susceptible Balb/c mice following intraperitoneal inoculation withSL1344lux.

FIG. 10A depicts an image prior to the opening of the peritaneal cavity.

FIG. 10B depicts an image after the opening of the peritoneal cavity.

FIG. 10C depicts an image after the cecum was pulled to the left side.

FIGS. 11A, 11B, 11C, 11D, and 11E depict the effects of ciprofloxacintreatment on bioluminescence from SL1344lux Salmonella inorally-inoculated mice.

FIG. 11A shows a graph of the relative bioluminescence intensity,measured from the abdominal area, as a function of time after initiationof treatment, for treated and untreated animals.

FIGS. 11B and 11D depict composite images of mice 8 days after oralinoculation with SL1344lux Salmonella , before treatment withciprofloxacin.

FIGS. 11C and 11E depict composite images of the same mice 5.5 hourseither following treatment (FIG. 11E) or control (no treatment: FIG.11C).

FIG. 12 depicts bioluminescence as a reporter for replication of HIV-1in culture. The gray scale image of the plates at 24h, 60h, 96h, and 7d,as indicated, is shown.

FIG. 13 depicts an assessment of the promoter activity in tissues oftransgenic mice containing a construct composed of the regulatoryportion of the HIV LTR (U3 region) upstream of the coding sequence ofthe firefly luciferase gene. NRE, negative response element; ENH,enhancer region, TAR-transactivation responsive element.

FIG. 14 depicts topical delivery of substrate to dermal cells intransgenic mice containing a construct composed of the regulatoryportion of the HIV LTR upstream of the coding sequence of the fireflyluciferase gene.

FIG. 15 depicts bioluminescence from induced ears as result of topicalluciferin delivery in transgenic mice containing a construct composed ofthe regulatory portion of the HIV LTR upstream of the coding sequence ofthe firefly luciferase gene.

FIG. 16 depicts unilateral induction of luciferase expression intransgenic mice; the left half of the shaved dorsal surface of the miceand the left ear were treated with DMSO to activate expression of theHIV-1 LTR; luciferin was applied topically over the entire surface ofthe back and both ears.

FIG. 17 depicts the detection of bioluminescence from internal tissuesin transgenic mice.

FIG. 18 depicts imaging of the abdomen of animals following laprotomydemonstrating signals to localize the origin of internalbioluminescence.

FIG. 19 depicts expression of the differential expression of HIV-LTR inneonatal transgenic mice.

V. DETAILED DESCRIPTION OF THE INVENTION

A. Definitions

Unless otherwise indicated, all terms used herein have the same meaningas they would to one skilled in the art of the present invention.

Opaque medium is used herein to refer to a medium that is“traditionally” opaque, not necessarily absolutely opaque. Accordingly,an opaque medium is defined as a medium that is commonly considered tobe neither transparent nor translucent, and includes items such as awood board, and flesh and skin of a mammal.

Luciferase, unless stated otherwise, includes prokaryotic and eukaryoticluciferases, as well as variants possessing varied or altered opticalproperties, such as luciferases that luminesce at wavelengths in the redrange.

Biocompatible entity is an entity that can be administered to a mammal.This includes pathogens which may be deleterious to the mammal. Inreference to an animal whose cells contain a transgene expressing alight-generating protein, biocompatible entity refers to thetransgene-containing cells comprising the mammal.

Light-generating is defined as capable of generating light through achemical reaction or through the absorption of radiation.

Light is defined herein, unless stated otherwise, as electromagneticradiation having a wavelength of between about 300 nm and about 1100 nm.

Spread of infection typically refers to the spreading and colonizationby a pathogen of host sites other than the initial infection site. Theterm can also include, however, growth in size and/or number of thepathogen at the initial infection site.

-   -   lux—prokaryotic genes associated with luciferase and photon        emission.    -   luc—eukaryotic genes associated with luciferase and photon        emission.

Promoter induction event refers to an event that results in the director indirect induction of a selected inducible promoter.

Heterologous gene refers to a gene which has been transfected into ahost organism. Typically, a heterologous gene refers to a gene that isnot originally derived from the transfected or transformed cells'genomic DNA.

Transgene refers to a heterologous gene which has been introduced,transiently or permanently, into the germ line or somatic cells of anorganism.

B. General Overview of the Invention

The present invention includes methods and compositions relating tonon-invasive imaging and/or detecting of light-emitting conjugates inmammalian subjects. The conjugates contain a biocompatible entity and alight-generating moiety. Biocompatible entities include, but are notlimited to, small molecules such as cyclic organic molecules;macromolecules such as proteins; microorganisms such as viruses,bacteria, yeast and fungi; eukaryotic cells; all types of pathogens andpathogenic substances; and particles such as beads and liposomes. Inanother aspect, biocompatible entities may be all or some of the cellsthat constitute the mammalian subject being imaged.

Light-emitting capability is conferred on the entities by theconjugation of a light-generating moiety. Such moieties includefluorescent molecules, fluorescent proteins, enzymatic reactions givingoff photons and luminescent substances, such as bioluminescent proteins.The conjugation may involve a chemical coupling step, geneticengineering of a fusion protein, or the transformation of a cell,microorganism or animal to express a bioluminescent protein. Forexample, in the case where the entities are the cells constituting themammalian subject being imaged, the light-generating moiety may be abioluminescent or fluorescent protein “conjugated” to the cells throughlocalized, promoter-controlled expression from a vector constructintroduced into the cells by having made a transgenic or chimericanimal.

Light-emitting conjugates are typically administered to a subject by anyof a variety of methods, allowed to localize within the subject, andimaged. Since the imaging, or measuring photon emission from thesubject, may last up to tens of minutes, the subject is usually, but notalways, immobilized during the imaging process.

Imaging of the light-emitting entities involves the use of aphotodetector capable of detecting extremely low levels oflight—typically single photon events—and integrating photon emissionuntil an image can be constructed. Examples of such sensitivephotodetectors include devices that intensify the single photon eventsbefore the events are detected by a camera, and cameras (cooled, forexample, with liquid nitrogen) that are capable of detecting singlephotons over the background noise inherent in a detection system.

Once a photon emission image is generated, it is typically superimposedon a “normal” reflected light image of the subject to provide a frame ofreference for the source of the emitted photons (i.e., localize thelight-emitting conjugates with respect to the subject). Such a“composite” image is then analyzed to determine the location and/oramount of a target in the subject.

The steps and embodiments outlined above are presented in greaterdetail, below.

C. Light-Emitting Entities

1. Light-Generating Moieties.

The light-generating moieties (LGMs), molecules or constructs useful inthe practice of the present invention may take any of a variety offorms, depending on the application. They share the characteristic thatthey are luminescent, that is, that they emit electromagnetic radiationin ultraviolet (UV), visible and/or infra-red (IR) from atoms ormolecules as a result of the transition of an electronically excitedstate to a lower energy state, usually the ground state.

Examples of light-generating moieties include photoluminescentmolecules, such as fluorescent molecules, chemiluminescent compounds,phosphorescent compounds, and bioluminescent compounds.

Two characteristics of LGMs that bear considerable relevance to thepresent invention are their size and their spectral properties. Both arediscussed in the context of specific types of light-generating moietiesdescribed below, following a general discussion of spectral properties.

Spectral Properties. An important aspect of the present invention is theselection of light-generating moieties that produce light capable ofpenetrating animal tissue such that it can be detected externally in anon-invasive manner. The ability of light to pass through a medium suchas animal tissue (composed mostly of water) is determined primarily bythe light's intensity and wavelength.

The more intense the light produced in a unit volume, the easier thelight will be to detect. The intensity of light produced in a unitvolume depends on the spectral characteristics of individual LGMs,discussed below, and on the concentration of those moieties in the unitvolume. Accordingly, conjugation schemes that place a high concentrationof LGMs in or on an entity (such as high-efficiency loading of aliposome or high-level expression of a bioluminescent protein in a cell)typically produce brighter light-emitting conjugates (LECs), which areeasier to detect through deeper layers of tissue, than schemes whichconjugate, for example, only a single LGM onto each entity.

A second factor governing the detectability of an LGM through a layer oftissue is the wavelength of the emitted light. Water may be used toapproximate the absorption characteristics of animal tissue, since mosttissues are composed primarily of water. It is well known that watertransmits longer-wavelength light (in the red range) more readily thanit does shorter wavelength light.

Accordingly, LGMs which emit light in the range of yellow to red(550-1100 nm) are typically preferable to LGMs which emit at shorterwavelengths. Several of the LGMs discussed below emit in this range.However, it will be noted, based on experiments performed in support ofthe present invention and presented below, that excellent results can beachieved in practicing the present invention with LGMs that emit in therange of 486 nm, despite the fact that this is not an optimal emissionwavelength. These results are possible, in part, due to the relativelyhigh concentration of LGMs (luciferase molecules) present in the LECs(transformed Salmonella cells) used in these experiments, and to the useof a sensitive detector. It will be understood that through the use ofLGMs with a more optimal emission wavelength, similar detection resultscan be obtained with LGEs having lower concentrations of the LGMs.

Fluorescence-based Moieties. Fluorescence is the luminescence of asubstance from a single electronically excited state, which is of veryshort duration after removal of the source of radiation. The wavelengthof the emitted fluorescence light is longer than that of the excitingillumination (Stokes' Law), because part of the exciting light isconverted into heat by the fluorescent molecule.

Because fluorescent molecules require input of light in order toluminesce, their use in the present invention may be more complicatedthan the use of bioluminescent molecules. Precautions are typicallytaken to shield the excitatory light so as not to contaminate thefluorescence photon signal being detected from the subject. Obviousprecautions include the placement of an excitation filter, such thatemployed in fluorescence microscope, at the radiation source. Anappropriately-selected excitation filter blocks the majority of photonshaving a wavelength similar to that of the photons emitted by thefluorescent moiety. Similarly a barrier filter is employed at thedetector to screen out most of the photons having wavelengths other thanthat of the fluorescence photons. Filters such as those described abovecan be obtained from a variety of commercial sources, including OmegaOptical, Inc. (Brattleboro, Vt.).

Alternatively, a laser producing high intensity light near theappropriate excitation wavelength, but not near the fluorescenceemission wavelength, can be used to excite the fluorescent moieties. Anx-y translation mechanism may be employed so that the laser can scan thesubject, for example, as in a confocal microscope.

As an additional precaution, the radiation source can be placed behindthe subject and shielded, such that the only radiation photons reachingthe site of the detector are those that pass all the way through thesubject. Furthermore, detectors may be selected that have a reducedsensitivity to wavelengths of light used to excite the fluorescentmoiety.

Through judicious application of the precautions above, the detection offluorescent LGMs according to methods of the present invention ispossible.

Fluorescent moieties include small fluorescent molecules, such asfluorescein, as well as fluorescent proteins, such as green fluorescentprotein (Chalfie, et al., 1994, Science 263:802-805., Morin andHastings, 1971, J. Cell. Physiol. 77:313) and lumazine and yellowfluorescent proteins (O'Kane, et al., 1991, PNAS 88:1100-1104, Daubner,et al., 1987, PNAS 84:8912-8916). In addition, certain colored proteinssuch as ferredoxin IV (Grabau, et al., 1991, J Biol Chem.266:3294-3299), whose fluorescence characteristics have not beenevaluated, may be fluorescent and thus applicable for use with thepresent invention. Ferredoxin IV is a particularly promising candidate,as it has a reddish color, indicating that it may fluoresce or reflectat a relatively long wavelength and produce light that is effective atpenetrating tissue. Furthermore, the molecule is small for a protein (95amino acids), and can thus be conjugated to entities with a minimalimpact on their function.

An advantage of small fluorescent molecules is that they are less likelyto interfere with the bioactivity of the entity to which they areattached than a would a larger light-generating moiety. In addition,commercially-available fluorescent molecules can be obtained with avariety of excitation and emission spectra that are suitable for usewith the present invention. For example, Molecular Probes (Eugene,Oreg.) sells a number of fluorophores, including Lucifer Yellow (abs. at428 nm, and emits at 535 nm) and Nile Red (abs. at 551 nm and emits at636 nm). Further, the molecules can be obtained derivatized with avariety of groups for use with various conjugation schemes (e.g., fromMolecular Probes).

Bioluminescence-Based Moieties. The subjects of chemiluminescence(luminescence as a result of a chemical reaction) and bioluminescence(visible luminescence from living organisms) have, in many aspects, beenthoroughly studied (e.g., Campbell, 1988, Chemiluminescence. Principlesand Applications in Biology and Medicine (Chichester, England: EllisHorwood Ltd. and VCH Verlagsgesellschaft mbH)). A brief summary ofsalient features follows.

Bioluminescent molecules are distinguished from fluorescent molecules inthat they do not require the input of radiative energy to emit light.Rather, bioluminescent molecules utilize chemical energy, such as ATP,to produce light. An advantage of bioluminescent moieties, as opposed tofluorescent moieties, is that there is virtually no background in thesignal. The only light detected is light that is produced by theexogenous bioluminescent moiety. In contrast, the light used to excite afluorescent molecule often results in the fluorescence of substancesother than the intended target. This is particularly true when the“background” is as complex as the internal environment of a livinganimal.

Several types of bioluminescent molecules are known. They include theluciferase family (e.g., Wood, et al., 1989, Science 244:700-702) andthe aequorin family (e.g., Prasher, et al., Biochem. 26:1326-1332).Members of the luciferase family have been identified in a variety ofprokaryotic and eukaryotic organisms. Luciferase and other enzymesinvolved in the prokaryotic luminescent (lux) systems, as well as thecorresponding lux genes, have been isolated from marine bacteria in theVibrio and Photobacterium genera and from terrestrial bacteria in theXenorhabdus genus.

An exemplary eukaryotic organism containing a luciferase system (luc) isthe North American firefly Photinus pyralis. Firefly luciferase has beenextensively studied, and is widely used in ATP assays. cDNAs encodingluciferases from Pyrophorus plagiophthalamus, another species of clickbeetle, have been cloned and expressed (Wood, et al., 1989, Science244:700-702). This beetle is unusual in that different members of thespecies emit bioluminescence of different colors. Four classes ofclones, having 95-99% homology with each other, were isolated. They emitlight at 546 nm (green), 560 nm (yellow-green), 578 nm (yellow) and 593nm (orange). The last class (593 nm) may be particularly advantageousfor use as a light-generating moiety with the present invention, becausethe emitted light has a wavelength that penetrates tissues more easilythan shorter wavelength light.

Luciferases, as well as aequorin-like molecules, require a source ofenergy, such as ATP, NAD(P)H, and the like, and a substrate, such asluciferin or coelentrizine and oxygen.

The substrate luciferin must be supplied to the luciferase enzyme inorder for it to luminesce. In those cases where a luciferase enzyme isintroduced as an expression product of a vector containing cDNA encodinga lux luciferase, a convenient method for providing luciferin is toexpress not only the luciferase but also the biosynthetic enzymes forthe synthesis of luciferin. In cells transformed with such a construct,oxygen is the only extrinsic requirement for bioluminescence. Such anapproach, detailed in Example 1, is employed to generate lux-transformedSalmonella, which are used in experiments performed in support of thepresent invention and detailed herein.

The plasmid construct, encoding the lux operon obtained from the soilbacterium Xenorhabdus luminescens (Frackman, et al., 1990, J. Bact.172:5767-5773), confers on transformed E coli the ability to emitphotons through the expression of the two subunits of the heterodimericluciferase and three accessory proteins (Frackman, et al., 1990).Optimal bioluminescence for E. Coli expressing the lux genes of X.luminescens is observed at 37° C. (Szittner and Meighen, 1990, J. Biol.Chem. 265:16581-16587, Xi, et al., 1991, J. Bact. 173:1399-1405) incontrast to the low temperature optima of luciferases from eukaryoticand other prokaryotic luminescent organisms (Campbell, 1988,Chemiluminescence. Principles and Applications in Biology and Medicine(Chichester, England: Ellis Horwood Ltd. and VCH VerlagsgesellschaftmbH)). The luciferase from X. luminescens, therefore, is well-suited foruse as a marker for studies in animals.

Luciferase vector constructs such as the one described above and inExample 1, can be adapted for use in transforming a variety of hostcells, including most bacteria, and many eukaryotic cells (lucconstructs). In addition, certain viruses, such as herpes virus andvaccinia virus, can be genetically-engineered to express luciferase. Forexample, Kovacs Sz. and Mettenlieter, 1991, J. Gen. Virol. 72:2999-3008,teach the stable expression of the gene encoding firefly luciferase in aherpes virus. Brasier and Ron, 1992, Meth. in Enzymol. 216:386-396,teach the use of luciferase gene constructs in mammalian cells.Luciferase expression from mammalian cells in culture has been studiedusing CCD imaging both macroscopically (Israel and Honigman, 1991, Gene104:139-145) and microscopically (Hooper, et al., 1990, J. Biolum. andChemilum. 5:123-130).

2. Entities

The invention includes entities which have been modified or conjugatedto include a light-generating moiety, construct or molecule, such asdescribed above. Such conjugated or modified entities are referred to aslight-emitting entities, light-emitting conjugates (LECs) or simplyconjugates. The entities themselves may take the form of, for example,molecules, macromolecules, particles, microorganisms, or cells. Themethods used to conjugate a light-generating moiety to an entity dependon the nature of the moiety and the entity. Exemplary conjugationmethods are discussed in the context of the entities described below.

Small molecules. Small molecule entities which may be useful in thepractice of the present invention include compounds which specificallyinteract with a pathogen or an endogenous ligand or receptor. Examplesof such molecules include, but are not limited to, drugs or therapeuticcompounds; toxins, such as those present in the venoms of poisonousorganisms, including certain species of spiders, snakes, scorpions,dinoflagellates, marine snails and bacteria; growth factors, such asNGF, PDGF, TGF and TNF; cytokines; and bioactive peptides.

The small molecules are preferably conjugated to light-generatingmoieties that interfere only minimally, if at all, with the bioactivityof the small molecule, such as small fluorescent molecules (describedabove). Conjugations are typically chemical in nature, and can beperformed by any of a variety of methods known to those skilled in theart.

The small molecule entity may be synthesized to contain alight-generating moiety, so that no formal conjugation procedure isnecessary. Alternatively, the small molecule entity may be synthesizedwith a reactive group that can react with the light generating moiety,or vice versa.

Small molecules conjugated to light-generating moieties of the presentinvention may be used either in animal models of human conditions ordiseases, or directly in human subjects to be treated. For example, asmall molecule which binds with high affinity to receptor expressed ontumor cells may be used in an animal model to localize and obtain sizeestimates of tumors, and to monitor changes in tumor growth ormetastasis following treatment with a putative therapeutic agent. Suchmolecules may also be used to monitor tumor characteristics, asdescribed above, in cancer patients.

Macromolecules. Macromolecules, such as polymers and biopolymers,constitute another example of entities useful in practicing the presentinvention. Exemplary macromolecules include antibodies, antibodyfragments, fusion proteins and certain vector constructs.

Antibodies or antibody fragments, purchased from commercial sources ormade by methods known in the art (Harlow, et al., 1988, Antibodies: ALaboratory Manual, Chapter 10, pg. 402, Cold Spring Harbor Press), canbe used to localize their antigen in a mammalian subject by conjugatingthe antibodies to a light-generating moiety, administering the conjugateto a subject by, for example, injection, allowing the conjugate tolocalize to the site of the antigen, and imaging the conjugate.

Antibodies and antibody fragments have several advantages for use asentities in the present invention. By their nature, they constitutetheir own targeting moieties. Further, their size makes them amenable toconjugation with several types of light-generating moieties, includingsmall fluorescent molecules and fluorescent and bioluminescent proteins,yet allows them to diffuse rapidly relative to, for example, cells orliposomes.

The light-generating moieties can be conjugated directly to theantibodies or fragments, or indirectly by using, for example, afluorescent secondary antibody. Direct conjugation can be accomplishedby standard chemical coupling of, for example, a fluorophore to theantibody or antibody fragment, or through genetic engineering. Chimeras,or fusion proteins can be constructed which contain an antibody orantibody fragment coupled to a fluorescent or bioluminescent protein.For example, Casadei, et al., 1990, PNAS 87:2047-2051, describe a methodof making a vector construct capable of expressing a fusion protein ofaequorin and an antibody gene in mammalian cells.

Conjugates containing antibodies can be used in a number of applicationsof the present invention. For example, a labeled antibody directedagainst E-selectin, which is expressed at sites of inflammation, can beused to localize the inflammation and to monitor the effects of putativeanti-inflammatory agents.

Vector constructs by themselves can also constitute macromolecularentities applicable to the present invention. For example, a eukaryoticexpression vector can be constructed which contains a therapeutic geneand a gene encoding a light-generating molecule under the control of aselected promoter (i.e., a promoter which is expressed in the cellstargeted by the therapeutic gene). Expression of the light-generatingmolecule, assayed using methods of the present invention, can be used todetermine the location and level of expression of the therapeutic gene.This approach may be particularly useful in cases where the expressionof the therapeutic gene has no immediate phenotype in the treatedindividual or animal model.

Viruses. Another entity useful for certain aspects of the invention areviruses. As many viruses are pathogens which infect mammalian hosts, theviruses may be conjugated to a light-generating moiety and used to studythe initial site and spread of infection. In addition, viruses labeledwith a light-generating moiety may be used to screen for drugs whichinhibit the infection or the spread of infection.

A virus may be labeled indirectly, either with an antibody conjugated toa light-generating moiety, or by, for example, biotinylating virions(e.g., by the method of Dhawan, et al., 1991, J. Immunol. 147(1):102)and then exposing them to streptavidin linked to a detectable moiety,such as a fluorescent molecule.

Alternatively, virions may be labeled directly with a fluorophore likerhodamine, using, for example, the methods of Fan, et al., 1992, J.Clin. Micro. 30(4):905. The virus can also be genetically engineered toexpress a light-generating protein. The genomes of certain viruses, suchas herpes and vaccinia, are large enough to accommodate genes as largeas the lux or luc genes used in experiments performed in support of thepresent invention.

Labeled virus can be used in animal models to localize and monitor theprogression of infection, as well as to screen for drugs effective toinhibit the spread of infection. For example, while herpes virusinfections are manifested as skin lesions, this virus can also causeherpes encephalitis. Such an infection can be localized and monitoredusing a virus labeled by any of the methods described above, and variousantiviral agents can be tested for efficacy in central nervous system(CNS) infections.

Particles. Particles, including beads, liposomes and the like,constitute another entity useful in the practice of the presentinvention. Due to their larger size, particles may be conjugated with alarger number of light-generating molecules than, for example, can smallmolecules. This results in a higher concentration of light emission,which can be detected using shorter exposures or through thicker layersof tissue. In addition, liposomes can be constructed to contain anessentially pure targeting moiety, or ligand, such as an antigen or anantibody, on their surface. Further, the liposomes may be loaded with,for example, bioluminescent protein molecules, to relatively highconcentrations (Campbell, 1988, Chemiluminescence. Principles andApplications in Biology and Medicine (Chichester, England: Ellis HorwoodLtd. and VCH Verlagsgesellschaft mbH)).

Furthermore, two types of liposomes may be targeted to the same celltype such that light is generated only when both are present. Forexample, one liposome may carry luciferase, while the other carriesluciferin. The liposomes may carry targeting moieties, and the targetingmoieties on the two liposomes may be the same or different. Viralproteins on infected cells can be used to identify infected tissues ororgans. Cells of the immune system can be localized using a single ormultiple cell surface markers.

The liposomes are preferably surface-coated, e.g., by incorporation ofphospholipid—polyethyleneglycol conjugates, to extend blood circulationtime and allow for greater targeting via the bloodstream. Liposomes ofthis type are well known.

Cells. Cells, both prokaryotic and eukaryotic, constitute another entityuseful in the practice of the present invention. Like particles, cellscan be loaded with relatively high concentrations of light-generatingmoieties, but have the advantage that the light-generating moieties canbe provided by, for example, a heterologous genetic construct used totransfect the cells. In addition, cells can be selected that express“targeting moieties”, or molecules effective to target them to desiredlocations within the subject. Alternatively, the cells can betransfected with a vector construct expressing an appropriate targetingmoiety.

The cell type used depends on the application. For example, as isdetailed below, bacterial cells, such as Salmonella, can be used tostudy the infective process, and to evaluate the effects of drugs ortherapeutic agents on the infective process with a high level oftemporal and spatial resolution.

Bacterial cells constitute effective entities. For example, they can beeasily transfected to express a high levels of a light-generatingmoiety, as well as high levels of a targeting protein. In addition, itis possible to obtain E. coli libraries containing bacteria expressingsurface-bound antibodies which can be screened to identify a colonyexpressing an antibody against a selected antigen (Stratagene, La Jolla,Calif.). Bacteria from this colony can then be transformed with a secondplasmid containing a gene for a light-generating protein, andtransformants can be utilized in the methods of the present invention,as described above, to localize the antigen in a mammalian host.

Pathogenic bacteria can be conjugated to a light-generating moiety andused in an animal model to follow the infection process in vivo and toevaluate potential anti-infective drugs, such as new antibiotics, fortheir efficacy in inhibiting the infection. An example of thisapplication is illustrated by experiments performed in support of thepresent invention and detailed below.

Eukaryotic cells are also useful as entities in aspects of the presentinvention. Appropriate expression vectors, containing desired regulatoryelements, are commercially available. The vectors can be used togenerate constructs capable of expressing desired light-generatingproteins in a variety of eukaryotic cells, including primary culturecells, somatic cells, lymphatic cells, etc. The cells can be used intransient expression studies, or, in the case of cell lines, can beselected for stable transformants.

Expression of the light-generating protein in transformed cells can beregulated using any of a variety of selected promoters. For example, ifthe cells are to be used as light-emitting entities targeted to a sitein the subject by an expressed ligand or receptor, aconstitutively-active promoter, such as the CMV or SV40 promoter may beused. Cells transformed with such a construct can also be used to assayfor compounds that inhibit light generation, for example, by killing thecells.

Alternatively, the transformed cells may be administered such theybecome uniformly distributed in the subject, and express thelight-generating protein only under certain conditions, such as uponinfection by a virus or stimulation by a cytokine. Promoters thatrespond to factors associated with these and other stimuli are known inthe art. In a related aspect, inducible promoters, such as the Tetsystem (Gossen and Bujard, 1992, PNAS 89:5547-5551) can be used totransiently activate expression of the light-generating protein.

For example, CD4+ lymphatic cells can be transformed with a constructcontaining tat-responsive HIV LTR elements, and used as an assay forinfection by HIV (Israel and Honigman, 1991, Gene 104:139-145). Cellstransformed with such a construct can be introduced into SCID-hu mice(McCune, et al., 1988, Science 241:1632-1639) and used as model forhuman HIV infection and AIDS.

Tumor cell lines transformed as above, for example, with aconstitutively-active promoter, may be used to monitor the growth andmetastasis of tumors. Transformed tumor cells may be injected into ananimal model, allowed to form a tumor mass, and the size and metastasisof the tumor mass monitored during treatment with putative growth ormetastasis inhibitors.

Tumor cells may also be generated from cells transformed with constructscontaining regulatable promoters, whose activity is sensitive to variousinfective agents, or to therapeutic compounds.

Cell Transformation. Transformation methods for both prokaryotic cellsand eukaryotic cells are well known in the art (Sambrook, et al., 1989,In Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Vol. 2). Vectors containing the appropriate regulatory elementsand multiple cloning sites are widely commercially available (e.g.,Stratagene, La Jolla, Calif., Clontech, Palo Alto, Calif.).

D. Transgenic Animals Containing Genes Encoding Light-GeneratingProteins

In another aspect, the present invention includes transgenic animalscontaining a heterologous gene construct encoding a light-generatingprotein or complex of proteins. The construct is driven by a selectedpromoter, and can include, for example, various accessory proteinsrequired for the functional expression of the light-generating protein,as well as selection markers and enhancer elements.

Activation of the promoter results in increased expression of the genesencoding the light-generating molecules and accessory proteins.Activation of the promoter is achieved by the interaction of a selectedbiocompatible entity, or parts of the entity, with the promoterelements. If the activation occurs only in a part of the animal, onlycells in that part will express the light-generating protein.

For example, an interferon-inducible promoter, such as the promoter for3′-5′ poly-A synthetase or the Mx protein (an interferon-induciblepromoter), can be used to detect the infection of transgenic cells by anumber of different RNA viruses.

In a related aspect, a promoter expressed in certain disease states canbe used to mark affected areas in a transgenic animal, and expression ofthe light-generating moiety can be used to monitor the effects oftreatments for the disease state. For example, E-selectin is expressedat sites of inflammation in vivo (Pober and Cotran, 1991, Lab. Invest.64:301-305). Accordingly, the E-selectin promoter can be isolated andused to drive the expression of a luciferase gene.

It is also possible to use methods of the invention with tissue-specificpromoters. This enables, for example, the screening of compounds whichare effective to inhibit pathogenic processes resulting in thedegeneration of a particular organ or tissue in the body, and permitsthe tracking of cells (e.g., neurons) in, for example, a developinganimal.

Many promoters which are applicable for use with the present inventionare known in the art. In addition, methods are known for isolatingpromoters of cloned genes, using information from the gene's cDNA toisolate promoter-containing genomic DNA.

In a specific embodiment of the present invention, transgenic animalsexpressing luciferase under the control of the HIV-1 LTR have beengenerated. As demonstrated in specific examples, luciferase expressionserves as a real-time bioluminescent reporter which allows thenoninvasive assessment of the level of promoter activity in vivo. Asdescribed, supra, the photons from the in vivo luciferase reaction canbe detected by a CCD camera, after transmission through animal tissues,and used as an indication of the level and location of gene expressionboth in superficial and internal tissues.

E. Imaging of Light-Emitting Conjugates

Light emitting conjugates that have localized to their intended sites ina subject may be imaged in a number of ways. Guidelines for suchimaging, as well as specific examples, are described below.

1. Localization of Light-Emitting Conjugates

In the case of “targeted” entities, that is, entities which contain atargeting moiety—a molecule or feature designed to localize the entitywithin a subject or animal at a particular site or sites, localizationrefers to a state when an equilibrium between bound, “localized”, andunbound, “free” entities within a subject has been essentially achieved.The rate at which such an equilibrium is achieved depends upon the routeof administration. For example, a conjugate administered by intravenousinjection to localize thrombi may achieve localization, or accumulationat the thrombi, within minutes of injection. On the other hand, aconjugate administered orally to localize an infection in the intestinemay take hours to achieve localization.

Alternatively, localization may simply refer to the location of theentity within the subject or animal at selected time periods after theentity is administered. For example, in experiments detailed herein,Salmonella are administered (e.g., orally) and their spread is followedas a function of time. In this case, the entity can be “localized”immediately following the oral introduction, inasmuch as it marks theinitial location of the administered bacteria, and its subsequent spreador recession (also “localization”) may be followed by imaging.

In a related aspect, localization of, for example, injected tumors cellsexpressing a light-generating moiety, may consist of the cellscolonizing a site within the animal and forming a tumor mass.

By way of another example, localization is achieved when an entitybecomes distributed following administration. For example, in the caseof a conjugate administered to measure the oxygen concentration invarious organs throughout the subject or animal, the conjugate becomes“localized”, or informative, when it has achieved an essentiallysteady-state of distribution in the subject or animal.

In all of the above cases, a reasonable estimate of the time to achievelocalization may be made by one skilled in the art. Furthermore, thestate of localization as a function of time may be followed by imagingthe light-emitting conjugate according to the methods of the invention.

2. Photodetector Devices

An important aspect of the present invention is the selection of aphotodetector device with a high enough sensitivity to enable theimaging of faint light from within a mammal in a reasonable amount oftime, preferably less than about 30 minutes, and to use the signal fromsuch a device to construct an image.

In cases where it is possible to use light-generating moieties which areextremely bright, and/or to detect light-emitting conjugates localizednear the surface of the subject or animal being imaged, a pair of“night-vision” goggles or a standard high-sensitivity video camera, suchas a Silicon Intensified Tube (SIT) camera (e.g., Hamamatsu PhotonicSystems, Bridgewater, N.J.), may be used. More typically, however, amore sensitive method of light detection is required.

In extremely low light levels, such as those encountered in the practiceof the present invention, the photon flux per unit area becomes so lowthat the scene being imaged no longer appears continuous. Instead, it isrepresented by individual photons which are both temporally andspatially distinct form one another. Viewed on a monitor, such an imageappears as scintillating points of light, each representing a singledetected photon.

By accumulating these detected photons in a digital image processor overtime, an image can be acquired and constructed. In contrast toconventional cameras where the signal at each image point is assigned anintensity value, in photon counting imaging the amplitude of the signalcarries no significance. The objective is to simply detect the presenceof a signal (photon) and to count the occurrence of the signal withrespect to its position over time.

At least two types of photodetector devices, described below, can detectindividual photons and generate a signal which can be analyzed by animage processor.

Reduced-Noise Photodetection Devices. The first class constitutesdevices which achieve sensitivity by reducing the background noise inthe photon detector, as opposed to amplifying the photon signal. Noiseis reduced primarily by cooling the detector array. The devices includecharge coupled device (CCD) cameras referred to as “backthinned”, cooledCCD cameras. In the more sensitive instruments, the cooling is achievedusing, for example, liquid nitrogen, which brings the temperature of theCCD array to approximately −120° C. The “backthinned” refers to anultra-thin backplate that reduces the path length that a photon followsto be detected, thereby increasing the quantum efficiency. Aparticularly sensitive backthinned cryogenic CCD camera is the “TECH512”, a series 200 camera available from Photometrics, Ltd. (Tucson,Ariz.).

Photon Amplification Devices. A second class of sensitive photodetectorsincludes devices which amplify photons before they hit the detectionscreen. This class includes CCD cameras with intensifiers, such asmicrochannel intensifiers. A microchannel intensifier typically containsa metal array of channels perpendicular to and co-extensive with thedetection screen of the camera. The microchannel array is placed betweenthe sample, subject, or animal to be imaged, and the camera. Most of thephotons entering the channels of the array contact a side of a channelbefore exiting. A voltage applied across the array results in therelease of many electrons from each photon collision. The electrons fromsuch a collision exit their channel of origin in a “shotgun” pattern,and are detected by the camera.

Even greater sensitivity can be achieved by placing intensifyingmicrochannel arrays in series, so that electrons generated in the firststage in turn result in an amplified signal of electrons at the secondstage. Increases in sensitivity, however, are achieved at the expense ofspatial resolution, which decreases with each additional stage ofamplification.

An exemplary microchannel intensifier-based single-photon detectiondevice is the C2400 series, available from Hamamatsu.

Image Processors. Signals generated by photodetector devices which countphotons need to be processed by an image processor in order to constructan image which can be, for example, displayed on a monitor or printed ona video printer. Such image processors are typically sold as part ofsystems which include the sensitive photon-counting cameras describedabove, and accordingly, are available from the same sources (e.g.,Photometrics, Ltd., and Hamamatsu). Image processors from other vendorscan also be used, but more effort is generally required to achieve afunctional system.

The image processors are usually connected to a personal computer, suchas an IBM-compatible PC or an Apple Macintosh (Apple Computer,Cupertino, Calif.), which may or may not be included as part of apurchased imaging system. Once the images are in the form of digitalfiles, they can be manipulated by a variety of image processing programs(such as “ADOBE PHOTOSHOP”, Adobe Systems, Adobe Systems, Mt. View,Calif.) and printed.

3. Immobilizing Subject in Detection Field of Device

Detection Field Of Device. The detection field of the device is definedas the area from which consistent measurements of photon emission can beobtained. In the case of a camera using an optical lens, the detectionfield is simply the field of view accorded to the camera by the lens.Similarly, if the photodetector device is a pair of “night vision”goggles, the detection field is the field of view of the goggles.

Alternatively, the detection field may be a surface defined by the endsof fiber-optic cables arranged in a tightly-packed array. The array isconstructed to maximize the area covered by the ends of the cables, asopposed to void space between cables, and placed in close proximity tothe subject. For instance, a clear material such as plexiglass can beplaced adjacent the subject, and the array fastened adjacent the clearmaterial, opposite from the subject.

The fiber-optic cable ends opposite the array can be connected directlyto the detection or intensifying device, such as the input end of amicrochannel intensifier, eliminating the need for a lens.

An advantage of this method is that scattering and/or loss of photons isreduced by eliminating a large part of the air space between the subjectand the detector, and/or by eliminating the lens. Even ahigh-transmission lens, such as the 60 mm AF Nikkor macro lens used inexperiments performed in support of the present invention, transmitsonly a fraction of the light reaching the front lens element.

With higher-intensity LGMs, photodiode arrays may be used to measurephoton emission. A photodiode array can be incorporated into arelatively flexible sheet, enabling the practitioner to partially “wrap”the array around the subject. This approach also minimizes photon loss,and in addition, provides a means of obtaining three-dimensional imagesof the bioluminescence.

Other approaches may be used to generate three-dimensional images,including multiple detectors placed around the subject or a scanningdetector or detectors.

It will be understood that the entire animal or subject need notnecessarily be in the detection field of the photodetection device. Forexample, if one is measuring a light-emitting conjugate known to belocalized in a particular region of the subject, only light from thatregion, and a sufficient surrounding “dark” zone, need be measured toobtain the desired information.

Immobilizing The Subject. In those cases where it is desired to generatea two-dimensional or three-dimensional image of the subject, the subjectmay be immobilized in the detection field of the photodetection devicesduring the period that photon emission is being measured. If the signalis sufficiently bright that an image can be constructed from photonemission measured in less than about 20 milliseconds, and the subject isnot particularly agitated, no special immobilization precautions may berequired, except to insure that the subject is in the field of thedetection device at the start of the measuring period.

If, on the other hand, the photon emission measurement takes longer thanabout 20 msec, and the subject is agitated, precautions to insureimmobilization of the subject during photon emission measurement,commensurate with the degree of agitation of the subject, need to beconsidered to preserve the spatial information in the constructed image.For example, in a case where the subject is a person and photon emissionmeasurement time is on the order of a few seconds, the subject maysimply be asked to remain as still as possible during photon emissionmeasurement (imaging). On the other hand, if the subject is an animal,such as a mouse, the subject can be immobilized using, for example, ananesthetic or a mechanical restraining device.

A variety of restraining devices may be constructed. For example, arestraining device effective to immobilize a mouse for tens of secondsto minutes may be built by fastening a plexiglass sheet over a foamcushion. The cushion has an indentation for the animal's head at oneend. The animal is placed under the plexiglass such that its head isover the indentation, allowing it to breathe freely, yet the movement ofits body is constrained by the foam cushion.

In cases where it is desired to measure only the total amount of lightemanating from a subject or animal, the subject does not necessarilyneed to be immobilized, even for long periods of photon emissionmeasurements. All that is required is that the subject be confined tothe detection field of the photodetector during imaging. It will beappreciated, however, that immobilizing the subject during suchmeasuring may improve the consistency of results obtained, because thethickness of tissue through which detected photons pass will be moreuniform from animal to animal.

4. Further Considerations During Imaging

Fluorescent Light-Generating Moieties. The visualization of fluorescentlight-generating moieties requires an excitation light source, as wellas a photodetector. Furthermore, it will be understood that theexcitation light source is turned on during the measuring of photonemission from the light-generating moiety.

Appropriate selection of a fluorophore, placement of the light sourceand selection and placement of filters, all of which facilitate theconstruction of an informative image, are discussed above, in thesection on fluorescent light-generating moieties.

High-Resolution Imaging. Photon scattering by tissue limits theresolution that can be obtained by imaging LGMs through a measurement oftotal photon emission. It will be understood that the present inventionalso includes embodiments in which the light-generation of LGMs issynchronized to an external source which can be focused at selectedpoints within the subject, but which does not scatter significantly intissue, allowing the construction of higher-resolution images. Forexample, a focused ultrasound signal can be used to scan, in threedimensions, the subject being imaged. Light-generation from areas whichare in the focal point of the ultrasound can be resolved from otherphoton emission by a characteristic oscillation imparted to the light bythe ultrasound (e.g., Houston and Moerner, U.S. Pat. No. 4,614,116,issued 30, Sep. 1986.)

5. Constructing an Image of Photon Emission

In cases where, due to an exceptionally bright light-generating moietyand/or localization of light-emitting conjugates near the surface of thesubject, a pair of “night-vision” goggles or a high sensitivity videocamera was used to obtain an image, the image is simply viewed ordisplayed on a video monitor. If desired, the signal from a video cameracan be diverted through an image processor, which can store individualvideo frames in memory for analysis or printing, and/or can digitize theimages for analysis and printing on a computer.

Alternatively, if a photon counting approach is used, the measurement ofphoton emission generates an array of numbers, representing the numberof photons detected at each pixel location, in the image processor.These numbers are used to generate an image, typically by normalizingthe photon counts (either to a fixed, pre-selected value, or to themaximum number detected in any pixel) and converting the normalizednumber to a brightness (greyscale) or to a color (pseudocolor) that isdisplayed on a monitor. In a pseudocolor representation, typical colorassignments are as follows. Pixels with zero photon counts are assignedblack, low counts blue, and increasing counts colors of increasingwavelength, on up to red for the highest photon count values. Thelocation of colors on the monitor represents the distribution of photonemission, and, accordingly, the location of light-emitting conjugates.

In order to provide a frame of reference for the conjugates, a greyscaleimage of the (still immobilized) subject from which photon emission wasmeasured is typically constructed. Such an image may be constructed, forexample, by opening a door to the imaging chamber, or box, in dim roomlight, and measuring reflected photons (typically for a fraction of thetime it takes to measure photon emission). The greyscale image may beconstructed either before measuring photon emission, or after.

The image of photon emission is typically superimposed on the greyscaleimage to produce a composite image of photon emission in relation to thesubject.

If it desired to follow the localization and/or the signal from alight-emitting conjugate over time, for example, to record the effectsof a treatment on the distribution and/or localization of a selectedbiocompatible moiety, the measurement of photon emission, or imaging canbe repeated at selected time intervals to construct a series of images.The intervals can be as short as minutes, or as long as days or weeks.

F. Analysis of Photon Emission Images

Images generated by methods and/or using compositions of the presentinvention may be analyzed by a variety of methods. They range from asimple visual examination, mental evaluation and/or printing of ahardcopy, to sophisticated digital image analysis. Interpretation of theinformation obtained from an analysis depends on the phenomenon underobservation and the entity being used.

The following experiments illustrate one application of the presentinvention—tracking Salmonella infection in live mice—and how imagesobtained using methods of the present invention can be analyzed.Similarily, infection of numerous other pathogens, including, but notlimited to, Pseudomonas, Staphylococcus, Streptococcus, Enterococcus,Enterobacter, Citrobacter, Leginella, Helicobacter, Acinetobacter,Escherichia, Klebsiella and Serratia.

G. Imaging of Luminescent Salmonella in Living Mice

Experiments performed in support of the present invention characterizethe distribution of Salmonella typhimurium infection in mice, the animalmodel of human typhoid. A mouse virulent Salmonella typhimurium strain,SL1344 (Hoiseth and Stocker, 1981, Nature 291:238-239), a non-invasivemutant of SL1344, BJ66 and a low virulence LT-2 strain of Salmonella ,LB5000 were each marked with a plasmid containing the lux operon, andused in experiments to localize Salmonella infection in mice.

1. Constructions of Luminescent Salmonella

Salmonella Strains. Three strains of Salmonella typhimurium withdiffering virulence phenotypes, defined by oral and intra-peritonealinoculations into mice, are selected for transformation.

The most virulent phenotype used herein is SL1344, a mouse strainoriginally obtained from a fatal infection of a calf (Hoiseth andStocker, 1981, Nature 291:238-239). Following oral inoculations of micewith this strain, bacteria are disseminated systematically via thelymphatic system resulting in colonization of the liver, spleen and bonemarrow (Carter and Collins, 1974, J. Exper. Med. 139:1189-1203.; seealso reviews by Finlay and Falkow, 1989, Mol. Microbiol. 3:1833-1841,and Hsu, 1989, Microbiol. Rev. 53:390-409.)

A non-invasive mutant of SL1344, BJ66, is also evaluated. Systemicinfections in mice do not typically result from, an oral inoculationwith BJ66, but do result from intraperitoneal inoculations with thisstrain.

A low virulence LT-2 strain of Salmonella , LB5000, is also examined.LT-2 stains are laboratory strains known to be of reduced or variablevirulence for mice. LB5000 contains multiple auxotrophic mutations, isstreptomycin resistant, and is cleared from mice following oral orintraperitoneal inoculations.

Transformation of Salmonella Strains with the lux Operon. The threestrains are each transformed with a plasmid encoding the lux operon, asdetailed in Example 1. The plasmid, obtained from the soil bacteriumXenorhabdus luminescens (Frackman, et al., 1990) confers on E coli theability to emit photons through the expression of the two subunits ofthe heterodimeric luciferase and three accessory proteins, luxC, luxDand luxE.

Inclusion of luxC, luxD and luxE removes the necessity of providing thefatty aldehyde substrate, luciferin, to the luciferase-expressing cells.Because supplying the substrate to eukaryotic luciferase enzymes in anin vivo system such as described herein may prove difficult, the entirelux operon of X. luminescens is used. The operon also encodes theenzymes for the biosynthesis of the fatty aldehyde substrate.

X. luminescens luciferase, an alpha-beta heterodimeric mixed-functionoxidase, catalyzes the oxidation of reduced flavin and long-chainaldehyde to oxidized flavin and the corresponding long-chain fatty acid.A fatty acid reductase complex is required for the generation andrecycling of fatty acid to aldehyde, and an NAD(P)H:flavinoxidoreductase supplies the reduced flavin.

Optimal bioluminescence for E. Coli expressing the lux genes of X.luminescens is 37° C. (Szittner and Meighen, 1990, J. Biol. Chem.265:16581-16587, Xi, et al., 1991, J. Bact. 173:1399-1405). In contrast,luciferases from eukaryotic and other prokaryotic luminescent organismstypically have lower temperature optima (Campbell, 1988,Chemiluminescence. Principles and Applications in Biology and Medicine(Chichester, England: Ellis Horwood Ltd. and VCH VerlagsgesellschaftmbH)). The luciferase from X. luminescens, therefore, is well-suited foruse as a marker for studies in animals.

The three strains are transformed by electroporation with the plasmidpGSL1, which contains the entire X. luminescens lux operon and confersresistance to ampicillin and carbenicillin on the Salmonella (Frackman,et al., 1990). The X. luminescens lux operon contains the genes luxA,luxb, luxC, luxD and luxE (Frackman, et al., 1990). LuxA and B encodethe two subunits of the heterodimeric luciferase. luxC and D encode thebiosynthetic enzymes for the luciferase substrate and luxE is aregulatory gene. Inclusion of the genes for the biosynthesis of thesubstrate is a convenient means of providing substrate to luciferase, incontrast to supplying luciferin externally to the cells in culture ortreating animals with the substrate.

2. Characterization of Transformed Salmonella in Vitro

Adherence And Invasive Properties. The adherence and invasive propertiesof the three Salmonella strains containing the lux plasmid are comparedin culture, to each other, and to their non-luminescent parental strainsby the standard invasion assay as described by Finlay and Falkow, 1989,Mol. Microbiol. 3:1833-1841., and detailed in Example 2.

In this assay, adherent and intracellular bacteria are quantifiedfollowing incubation with an epithelial cell line and peritonealmacrophages. The adherent and intracellular bacteria are detected andquantified by both the emission of photons from living cells, and colonyforming units following lysis and plating the cell lysates oncarbenicillin-containing plates.

The results of some of the assays are shown in FIGS. 2A through 2E anddiscussed in Example 8. The phenotypes of the three strains transformedwith the lux expressing plasmid are not significantly altered incomparison to the parental Salmonella strains. In addition, there is agood correlation between the intensity of bioluminescence and the CFUfrom the HEp-2 cells and macrophages. The results show thatluminescence, as an indicator of intracellular bacteria, is a rapidmethod for assaying the invasive properties of bacteria in culture.

BJ66 demonstrated reduced adherence to HEp-2 cells in comparison toSL1344, however, adherence of the two strains in primary cultures ofmurine peritoneal macrophages were comparable.

Light Emission. To evaluate the oxygen requirements of the system, 10fold serial dilutions of bacteria are placed in glass capillary tubesand imaged, as detailed in Example 3.

FIG. 3 shows an image generated in one such experiment. Luminescence isonly detected at the air-liquid interface, even in the tubes with smallnumbers of bacteria in air saturated medium (0.1 ml of air saturatedbuffer in 5 l results in a final O₂ concentration of 5 nM).

From these results, it is apparent that oxygen is likely a limitingfactor for luminescence.

Light Transmission Through Animal Tissue. To determine the degree towhich light penetrates animal tissue, light emitted from luminescentSalmonella and transmitted through tissue is quantified using ascintillation counter, with the fast coincidence detector turned off todetect single photons. The background due to dark current of thephotomultiplier tubes in this type of detection is significant, limitingthe assay to samples with relatively strong photon emission.

Four tissue types of varying opacity are compared using this approach:muscle from chicken breast, skin from chicken breast, lamb kidney andrenal medulla from lamb kidney. The number of photons that can bedetected through tissue is approximately ten fold less than the controlswithout tissue.

3. Characterization of Lux Salmonella in vivo

Oral Administration. Oral inoculation is natural route of infection ofmice or humans with Salmonella and results in a more protracted courseof disease. In order to study the progression of the Salmonellainfection following this route of inoculation, two strains of mice areinfected with the three strains of Salmonella . The results obtainedusing the resistant animals are discussed under the heading “Infectionof Resistant Mice”, below.

Balb/c mice are orally infected with suspensions of virulent SL1344lux,non-invasive BJ66lux and low virulence LB5000lux Salmonella , asdescribed in Example 5. Progression of the infection is followed byexternal imaging (Materials and Methods) over an 8 day period.

Representative images are shown in FIGS. 6A, 6B, and 6C. At 24 hourspost inoculation (p.i.), the bioluminescent signal is localized at asingle focus in all infected animals (FIGS. 6A, 6B and 6C).Bioluminescence disappears in all animals infected with the lowvirulence LB5000lux by 7 days p.i. (FIG. 6A). Animals infected with thevirulent SL1344lux, on the other hand, show virulent infection whichoften spreads over much of the abdominal cavity (FIG. 6C), though thetime at which it begins to spread is highly variable from animal toanimal. The infection by BJ66lux typically persists and remainslocalized at a single site (FIG. 6B).

I.P. Inoculation. To assess whether or not there is sufficient O₂ at thesites of Salmonella replication for the oxidation of luciferin andsubsequent luminescence (Campbell, 1988, Chemiluminescence. Principlesand Applications in Biology and Medicine (Chichester, England: EllisHorwood Ltd. and VCH Verlagsgesellschaft mbH)), photon emission ismeasured from the tissues of a respiring animal. Luminescent SL1344luxand LB5000lux are inoculated into the peritoneal cavities of two groupsof Balb/c mice. 32 hours post inoculation (p.i.), the transmittedphotons are imaged (FIG. 7).

In the mice infected with SL1344lux (left part of FIGURE), transmittedphotons are evident over a large surface, with foci of varyingintensities visible. These images are indicative of a disseminatedinfection, and are consistent with widespread colonization of theviscera, possibly including the liver and mesenteric lymph nodes. Incontrast, the distributions of transmitted photons from animals infectedwith the LB5000lux strain is very limited, indicating a limitedinfection.

The LB5000lux-infected mice remained healthy for several weeks p.i.,while the SL1344lux-infected mice were nearly moribund and euthanized at4 days p.i.

These experiments indicate that the level of O₂ in the blood and ortissues is adequate for bioluminescence of lux luciferase expressed bySalmonella . Furthermore, the experiments are consistent with theinvasive nature of the virulent strain SL1344 in comparison to thereduced virulent laboratory strain LB5000.

Infection Of Resistant Mice. Mice which are heterozygous at the Itylocus (Ity^(r/s)) are resistant to systemic infections by S. typhimurium(Plant and Glynn, 1976, J. Infect. Dis. 133:72-78). This locus, alsocalled Bcg (Gros, et al., 1981, J Immunol. 127:2417-2421) or Lsh(Bradley, 1977, Clin. and Exper. Immunol. 30:130-140), regulates thepathogenic processes of certain intracellular pathogens, such asMycobacterium lepraemurium (Forget, et al., 1981, Infect. Immunol.32:42-47), M. Bovis (Skamene, et al., 1984, Immunogenet. 19:117-120,Skamene and Pietrangeli, 1991, Nature 297:506-509) and M. intracelluare(Goto, et al., 1989, Immunogenetics 30:218-221). An analogous geneticcontrol of resistance and susceptibility to intracellular pathogensappears to be in humans as well (M. tuberculosis (Stead, 1992, Annals ofIntern. Med. 116:937-941, Stead, et al., et al., 1990, New Eng. J. Med.322:422-427) and M. leprae).

The Ity locus is located on mouse chromosome 1 with two allelic forms,Ity^(r) (resistant, dominant) and Ity^(s) (sensitive, recessive). Thegene encoded at the Ity locus apparently affects the ability ofmacrophages to disrupt the internalized pathogens (reviewed byBlackwell, et al., 1991, Immunol. Lett. 30:241-248 (1991); see alsoSkamene, et al., 1984, Immunogenet. 19:117-120, Skamene and Pietrangeli,1991, Nature 297:506-509) which in turn, affects the down streamfunction of the proposed macrophage-mediated transport of pathogens toother sites within the infected host. Balb/c mice are Ity^(s/s) and 129mice are Ity^(r/r). The heterozygous Balb/c×129 mice (Ity^(r/s)) areused in experiments detailed herein.

Resistant 129×Balb/c (Ity^(r/s)) viable mice are infected byintragastric inoculation of 1×10⁷ SL1344lux Salmonella as detailed inExample 7. The animals are imaged daily for 8 days post injection(d.p.i.).

Results are shown in FIGS. 8A (day 1) and 8B (day 8). The luminescence,detected by external imaging, is apparent at 24 h p.i., and appeared tolocalized to a single site in all animals. The luminescent signal ispresent throughout the study period (up to 8 days p.i.). The intensityof the luminescence and the location of the luminescent source issomewhat variable over time within a mouse and also from mouse to mouse.The luminescent tissue in all infected animals is the cecum (see below)and the variability in localization, and possibly intensity, is mostlikely due fact that internal organs of rodents are not tightly fixed inposition.

The apparent limited infection observed in these animals supports theinterpretation that the Ity restriction blocks macrophage transport. Thepersistence of this infection for 10 days, however, suggests that thereis adherence to the intestine mucosa and prolonged shedding of bacteriain the feces of these animals, as evidenced by luminescent fecalpellets. These results indicate that the luminescent phenotype of theSalmonella in vivo is retained over an 8 day duration in Ity restrictedanimals and that localization is possible following an oral inoculation.

Internal Imaging Following Oral Inoculation. In order to furtherlocalize the luminescent signal in the abdominal cavity, infected miceare imaged following laparotomy (Example 8). The predominant diseasemanifestation in all of the animals infected by the oral route is anenlarged cecum (FIGS. 9A, 9B, 9C). The “external” image (FIG. 9A)illustrates a focal luminescence, which is revealed in thepost-laparotomy image (FIG. 9B) to be the cecum.

Injection of air into the intestine confirms the presence of bacteria inother regions of the digestive tract. Bacteria in the colon and rectumare likely expressing luciferase, but low oxygen concentrations arelikely limiting light emission from these sites.

The images obtained from oral inoculation studies indicate that theluminescent signal, at 2 days p.i. and at 7 days p.i., localizes almostentirely to the cecum in each of the animals (Popesko, et al., 1990, AColour Atlas of Anatomy of Small Laboratory Animals Vol. Two: Rat MouseHamster (London England: Wolfe)) except those infected with LB5000lux.Luminescence is also apparent in the colon in some animals. By 7 daysp.i., no luminescence is detectable in the LB5000lux-infected animals.The CFU present in the organs of these mice are determined at 2 and 5 dp.i.

In animals infected intragastrically with the invasive strain,SL1344lux, the luminescence in the cecum appears early and precedes asystemic infection. In contrast, infections with the non-invasiveBJ66lux strain result in a persistent luminescence from the cecum thatremains, in some animals, for the entire course of the study (8 days).By 8 days p.i., luminescence is detected over much of the abdominalsurface, resembling the distribution of photons following an i.p.inoculation, in the SL1344lux infected mice.

Infections with SL1344lux appear to become systemic, as predicted, withprogressively more photons being emitted from an increasing surfacearea. Luminescence appears to localize over the abdomen in infectionswith all strains with little detectable luminescence from outside thisarea. A large number of transmitted photons are localized as a singlefocus over the abdomen suggesting that even though the infection may besystemic, the greatest amount of replication may be in areas surroundingthe intestine.

Localization of the luminescence over the cecum indicates that not onlyare there large numbers of organisms in this region of the intestine,but also suggests that the Salmonella associate with cells of the mucosasuch that they can obtain sufficient oxygen for luminescence. Emissionof photons from luciferase is oxygen dependent and the expected oxygenlevels in the lumen of the cecum, or intestine in general, are below thelevels required for luminescence. The luciferase reaction is notexpected to be functional in the intestine unless the bacteria canobtain oxygen from cells of the intestinal epithelium.

Thus, the systemic infection seems to be related to the invasivephenotype and not to simply adherence to epithelial cells of theintestine. These experiments implicate the cecum in some role in thepathogenic process either in the carrier state or as a site ofdissemination.

Monitoring the progression of infections to different tissues maygreatly enhance the ability to understand these steps in the pathogenicprocess, and enable, the screening for compounds effective to inhibitthe pathogen at selected steps.

Internal Imaging Following I.P. Inoculation. Mice infectedintraperitoneally with SL1344lux are imaged before and after laparotomy(Example 9). The results are shown in FIG. 10. The images demonstrateluminescence over a majority of the abdomen with multiple foci oftransmitted photons. The cecum does not appear to contain luminescentSalmonella. The results from these experiments indicate that all strainsof Salmonella have sufficient O₂ to be luminescent in the early phasesof infection. However, entry of Salmonella into cells of the mucosa andsubsequent systemic infection is likely limited to strains with theinvasive phenotype, since systemic infections at later time points areonly apparent in SL1344lux-infected mice.

Effects Of Ciprofloxacin On Salmonella Infection. Experiments, detailedin Example 10, are performed to demonstrate that non-invasive imaging isuseful for following the response of an infection to drugs. Mice areorally inoculated with SL1344lux and treated with 100 mg ofciprofloxacin, an antibiotic effective against Salmonella infections.The mice are imaged at selected time periods following treatment, andthe extent of infection is quantitated by measuring photon emission.Photon emission in treated mice is compared to values before theinitiation of treatment, and to values from control mice that had beeninfected, but not treated. Results from one such experiment are shown inFIGS. 11A, 11B, 11C, 11D, and 11E and discussed in Example 10. Infectionis significantly reduced in mice treated with the antibiotic, comparedboth to the levels of pathogen at time zero in treated animals, and tolevels of pathogen in control animals throughout the treatment period.

Effects Of Carbenenicillin Selection. Ducluzeau, et al., 1970, Zeut.Bakt. 5313:533-548., demonstrated that treatment of animals withantibiotics facilitated colonization of the cecum with Salmonella . Themice in the present experiments are maintained on an antibiotic regimeof intramuscular injections of carbenicillin for the purpose ofselecting the Amp^(r) Salmonella containing the luciferase clone. Thistreatment may alter the course of the gastrointestinal infection, butthe observation that Salmonella can associate with the cells lining thececum indicates that oxygen is available for luminescence. Thisobservation is notable, since the lumen of the cecum is commonly thoughtto be an anaerobic environment.

H. Applications

The bioluminescence technology is broadly applicable to a variety ofhostpathogen systems and may also enable temporal and spatial evaluationof other biological events, as for example tumor progression and geneexpression in living mammals, and have application in pharmaceuticaldevelopment and screening. Widespread use of in vivo imaging ofpathogens may reduce the numbers of animals and time needed forexperiments pertaining to pathogenesis and/or the real-time studyantimicrobial agents. Furthermore, bioluminescent organisms may beuseful as biosensors in the living animal, much as luminescent bacteriaare used in environmental analyses. Korpela et al., for example,demonstrate that the limited oxygen supply in the lumen of the G.I.tract restricted bioluminescence to sites in which oxygen is accessibleto the Salmonella , perhaps directly from epithelial or other celltypes. Korpela, et al., 1989, J. Biolum. Chemilum. 4:551-554. Thisoxygen requirement may find utility as an indicator of intimatecell-cell interactions, or as a biosensor for studying oxygenconcentrations at various sites in living animals. In the following,several exemplary applications of this technology are described for thepurpose of illustration, but are in no way intended to limit the presentinvention.

1. Determination of Oxygen Levels

The oxygen requirement for luminescence of luciferase evidenced in theexperiments summarized above indicates that the present invention may beapplicable as a method of determining spatial gradients of oxygenconcentration in a subject. Luminescent bacteria have been used tomeasure oxygen levels in the range of 10-1 mM. The studies predict that0.1 nM is the lower limit of detection (Campbell, 1988,Chemiluminescence. Principles and Applications in Biology and Medicine(Chichester, England: Ellis Horwood Ltd. and VCH VerlagsgesellschaftmbH)). The imaging methods described herein may be used for studyingoxygen levels at various sites in living animals. For example,microorganisms that have been engineered to emit light in an O₂ orCa²⁺-dependent manner could be used as biosensors in a subject, muchlike luminescent bacteria are used in environmental analyses (Guzzo, etal., 1992, Tox. Lett. 64/65:687-693, Korpela, et al., 1989, J. Biolum.Chemilum. 4:551-554, Jassim, et al., 1990, J. Biolum. Chemilum.5:115-122). The dynamic range of luminescence with respect to O₂concentration is much broader and reaches lower O₂ concentrations thanO₂ probes (Campbell, 1988, Chemiluminescence. Principles andApplications in Biology and Medicine (Chichester, England: Ellis HorwoodLtd. and VCH Verlagsgesellschaft mbH)). Moreover, light emission inproportion to O₂ concentration is linear over a range of 30 nM to 8 mM,and 9 mM O₂ is required for ½ maximal luminescence.

2. Localization of Tumor Cells

The growth and metastatic spread of tumors in a subject may be monitoredusing methods and compositions of the present invention. In particular,in cases where an individual is diagnosed with a primary tumor, LECsdirected against the cells of the tumor can be used to both define theboundaries of the tumor, and to determine whether cells from the primarytumor mass have migrated and colonized distal sites.

For example, LECs, such as liposomes containing antibodies directedagainst tumor antigens and loaded with LGMs, can be administered to asubject, allowed to bind to tumor cells in the subject, imaged, and theareas of photon emission can be correlated with areas of tumor cells.

In a related aspect, images utilizing tumor-localizing LECs, such asthose described above, may be generated at selected time intervals tomonitor tumor growth, progression and metastasis in a subject over time.Such monitoring may be useful to record results of anti-tumor therapy,or as part of a screen of putative therapeutic compounds useful ininhibiting tumor growth or metastasis.

Alternatively, tumor cells can be transformed, transduced, transientlyor permanently, or otherwise made to emit light, with a luciferaseconstruct under the control of a constitutively-active promoter, andused to induce luminescent tumors in animal models, as described above.Such animal models can be used for evaluating the effects of putativeanti-tumor compounds.

3. Localization of Inflammation

In an analogous manner to that described above, compositions and methodsof the present invention may be used to localize sites of inflammation,monitor inflammation over time, and/or screen for effectiveanti-inflammatory compounds. Molecules useful for targeting to sites ofinflammation include the ELAN family of proteins, which bind toselections. An ELAN molecule can be incorporated as a targeting moietyon an entity of the present invention, and used to target inflammationsites.

Alternatively, an animal model for the study of putativeanti-inflammatory substances can be made by making the animal transgenicfor luciferase under the control of the E-selectin promoter. SinceE-selectin is expressed at sites of inflammation, transgenic cells atsites of inflammation would express luciferase.

The system can be used to screen for anti-inflammatory substances.Inflammatory stimuli can be administered to control and experimentalanimals, and the effects of putative anti-inflammatory compoundsevaluated by their effects on induced luminescence in treated animalsrelative to control animals.

4. Localization of Infection

As illustrated in experiments performed in support of the presentinvention and summarized above, LGCs may be effectively used to followthe course of infection of a subject by a pathogen, including, but notlimited to, Pseudomonas, Staphylococcus, Streptococcus, Enterococcus,Enterobacter, Citrobacter, Leginella, Helicobacter, Acinetobacter,Escherichia, Klebsiella or Serratia. In experiments detailed herein, theLGCs are pathogenic cells (Salmonella) transformed to expressluciferase. Such a system is ideally-suited to the study of infection,and the subsequent spread of infection, in animal models of humandiseases. It provides the ability to monitor the progression of aninfectious disease using sites of infection and disease progressionrather than traditional systemic symptoms, such as fever, swelling, etc.in studies of pathogenesis.

Use of an external imaging method to monitor the efficacy ofanti-infectives permits temporal and spatial evaluations in individualliving animals, thereby reducing the number of animals needed forexperiments pertaining to pathogenesis and/or the study anti-infectiveagents.

5. Monitoring Promoter Activity in Transgenic Mice

The generation of transgenic animals has become an important tool inbasic research and in the development of gene therapies and genevaccines. The present invention provides methods for rapid in situassessment of the uptake of nucleic acids and their expression and thusthe evaluation of gene delivery systems and DNA-based therapies.

More specifically, luciferase expression may serve as a real-timebioluminescent reporter, allowing the noninvasive assessment of thelevel of promoter activity in living animals. Photons from the in vivoluciferase reaction in the transgenic animal are detected by a CCDcamera, after transmission through animal tissues, and used as anindication of the level and location of gene expression. This way, areal-time assessment of the extent of promoter activity in bothsuperficial and deep tissues can be accomplished.

As described in specific embodiments of the present invention, thelight-emitting reporter systems in transgenic animals facilitate in vivoassessment of the regulation of gene expression, thus facilitating thedevelopment of novel therapies that target regulation of viral and hostgene expression. Bioluminescent reporters offer the advantages ofspontaneous emission of light without a need for outside light sources,low background signal permitting near single-event detection, real-timeanalyses, and the absence of cytotoxic photosensitizing dyes. As such,bioluminescent reporters have a greater versatility than fluorescentmarkers in mammalian tissues. Biological processes can be viewed in vivoby illuminating the temporal and spatial distribution of gene expressionin animals and humans.

The in vivo monitoring of promoter activity as described herein can beused for the assessment of gene delivery and expression in genetherapies, gene vaccines, antisense oligonucleotide therapies, thegeneration of chimeric and transgenic animals in research. Thetechnology is further useful for real-time noninvasive assays for geneexpression in research environments involving questions of developmentalregulation, response to infectious disease or other systems where geneexpression demonstrates change.

The following examples are given to enable those skilled in the art tomore clearly understand and to practice the present invention. Thepresent invention is not limited in scope by the exemplifiedembodiments, which are intended as illustrations of single aspects ofthe invention only, and methods which are functionally equivalent arewithin the scope of the invention. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingdrawings. Such modifications are intended to fall within the scope ofthe appended claims. The present invention is explained in more detailby means of the below examples.

VI. EXAMPLES

A. Materials and Methods

1. Cells

Salmonella strains SL1344 and LB5000 were obtained from B. A. D. Stocker(Stanford University; Hoiseth and Stocker, 1981, Nature 291:238-239).Salmonella strain BJ66 was obtained from B. D. Jones (StanfordUniversity).

HEp-2 cells were obtained from the American Type Culture Collection(ATCC; 12301 Parklawn Dr., Rockville Md.; Accession number CCL-23).

Murine peritoneal macrophages were obtained by peritoneal lavage ofeuthanized Balb/c mice with 7 ml of growth medium (Maximow and Bloom,1931, Textbook of Histology, Saunders, Philadelphia.)

2. Static Cultures

Low oxygen (static) cultures were prepared by inoculating 3 ml of LBBroth containing 100 mg/ml of carbenicillin with 6 μl of a bacterialsuspension from a stationary phase culture, and growing the bacteria at37° C. overnight in a stationary 7 ml culture tube.

3. Mice

Balb/c (Ity^(s/s)) mice were obtained from the Department of Oncology,Stanford University. 129×Balb/c (Ity^(r/s)) mice were obtained from theStanford Transgenic Animal Facility (Stanford, Calif.). All animals werehoused under identical conditions of photo period, feeding regime andtemperature in the Stanford University Research Animal Facility(Stanford, Calif.).

Anesthesia was performed by injecting the animals intraperitoneally(i.p.) with 33 μg/kg body weight nembutal.

Euthanasia was performed by asphyxiation in CO₂ or cervical dislocation,following protocols recommended by the Stanford University ResearchAnimal Facility. Cervical dislocation was used in experiments in whichresults may have been affected by physiological changes due to asphyxia.

Mice infected with lux-transformed Salmonella were given dailyintramuscular (i.m.) injections of carbenicillin (125 mg per kg bodyweight) to maintain selective pressure on the luminescent Salmonella forretention of the Amp^(r) plasmid containing the lux operon.

4. Imaging

Animals or objects to be imaged were immobilized in a light-tight boxcontaining a door and a charge-coupled device (CCD) camera with a twostage microchannel intensifier head (model C2400-40, Hamamatsu). Thecamera was attached, via cables leading out of the box, to an “ARGUS 50”image processor (Hamamatsu).

The ICCD system described above is capable of detecting single photonsonce a threshold of 10-30 photons is achieved. The signal to noise ratioof the system ranged from 2:1 to 1×10⁴:1, depending on signal intensity.

Grey-scale images were obtained by opening the light box door in dimroom light and integrating for 8-64 frames. The gain for the gray scaleimages was set to optimize the image—typically at 3000 volts on a scaleof 0 to 10,000 volts.

Bioluminescence data were obtained in absence of external illumination.Exposure settings were as follows: the black level was set automaticallyby the camera/image processor, the gain was adjusted automatically bythe intensifier controller, and the f-stop was set at 2.8. A 60 mm “AFNIKKOR” macro lens was used (Nikon Inc., Melville, N.Y.).

Bioluminescence images were generated by integrating photons for aselected period of time, typically 5 minutes. Data are presented at thelowest bit range setting of 0-3 bits per pixel for all animals. Forimages of other objects, i.e., 24 well plates, where the resolution ofthe bioluminescent signals was not possible at a bit range of 0-3, therange was increased to a setting that permitted localization ofbioluminescent signals, typically 1-7. Objects were imaged for shorterperiods of time when additional information could not be obtained byimaging for five minutes.

External imaging refers to non-invasive imaging of animals. Internalimaging refers to imaging after a partial dissection of the animals,typically a laparotomy. Internal imaging is performed in selectedanimals to confirm the sources of photon emission localized by externalimaging.

The bioluminescence image data are presented as a pseudo-colorluminescence image representing the intensity of the detected photons.Six levels of intensity are typically used, ranging from blue (lowintensity) to red (higher intensity).

To generate the FIGURES presented herein, greyscale and bioluminescenceimages were superimposed, using the image processor, to form a compositeimage providing a spatial frame of reference.

The composite image was displayed on an RGB CRT (red, green, blue;cathode ray tube) monitor, and the monitor was photographed to producehardcopies. Hardcopies were also generated by saving the image processorimage as a digital file, transferring the file to a computer, andprinting it on a color printer attached to the computer. Alternatively,hardcopies may be generated by printing the video signal directly usinga video printer.

B. Example 1 Transformation of Salmonella with pCGLS1 lux Plasmid

Salmonella strains SL1344, BJ66 and LB5000 were transformed with pCGLS1,a pUC18-based vector encoding the lux operon from Xenorhabdusluminescens (Frackman, et al., 1990).

1. pCGLS1 Plasmid

A schematic of the pCGLS1 plasmid is shown in FIGS. 1A, 1B and 1C. Theplasmid was constructed by cloning an ˜11 kb region encoding the luxgenes from the soil bacterium Xenorhabdus luminescens (FIG. 1A;Frackman, et al., 1990) into the Bam HI site (FIG. 1B) of pUC18 (FIG.1C; Clontech, Palo Alto, Calif.). The construction of the vector isdescribed by Frackman, et al., (1990).

Restriction enzyme sites in FIG. 1A are represented as follows: Bs, BstEII; C, Cla I; E, Eco RI; H, Hind III; M, Mlu I; S, Sca I; X, Xba I;B/Sa, Bam HI and Sau 3A junction. A sequence included in the multiplecloning site (MCS) is provided in FIG. 1B, with the Bam HI siteindicated in bold type.

A graphical representation of a pUC18 vector with no insert is shown inFIG. 1C. Labeled elements include an ampicillin resistance gene (Ap), alac Z gene (lac Z) and an E. coli origin of replication (Ori). Theunmodified pUC18 vector is approximately 2.7 kb in size.

2. Transformation of Salmonella

Electrocompetent cells from Salmonella strains SL1344, BJ66 and LB5000were made using standard methods (Sambrook, et al., 1989, In MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Vol.2) and stored at −80° C. until just prior to use. Electroporation wasperformed as follows: 1 μl of the plasmid (0.2 μg/ml) was added to 40 μlof ice-cold electrocompetent cells suspended in 10% glycerol. Thesuspension was mixed gently for one minute, placed in a 1 mm gapelectroporation cuvette and electroporated using a Bio-Rad Gene-Pulser(Bio-Rad Laboratories, Hercules, Calif.). The settings were 2.5 kvolts,400 ohms and 25 μfarads.

Following a one hour agitated incubation in Luria Bertini (LB) broth at37° C., the cells were plated on (LB) Agar containing 100 μg/mlcarbenicillin and allowed to grow overnight.

To maximize the bioluminescence of the labelled Salmonella, the luxoperon was maintained on a high-copy-number plasmid and not integratedas a single copy gene. However, plasmids are subject to modification bythe bacterial cell especially in recA strains, such as SL1344 and BJ66used in this study. The recA locus encodes a recombinase that may deleteregions of the plasmid containing the lux operon and the β-lactamase.Therefore, Salmonella recovered from cells in culture were plated bothin the presence or absence of carbenicillin, and were imaged todetermine the frequency at which bioluminescence was lost. All coloniesrecovered from gentamicin-treated, lysed HEp-2 cells and macrophageswere ampicillin resistant (Amp^(r)) and bioluminescent. Therefore, luxgenes appeared not to be lost during co-culture with mammalian cells.

Colonies were assayed for luminescence by visual inspection in a darkroom. Five transformants were identified as having high levels ofluminescence. Three of these, one each from the SL1344, BJ66 and LB5000strains, were selected for subsequent experiments. They were termedSL1344lux, BJ66lux and LB5000lux, respectively.

C. Example 2 Invasive Potential of Normal and Transformed Salmonella

The invasive potential of six strains of Salmonella (SL1344lux,LB5000lux, BJ66lux, SL1344, LB5000 and BJ66) was determined using twotypes of bacterial adherence and entry assays. Colony-forming units(CFU) assays were performed essentially as previously described (Finlayand Falkow, 1989, Mol. Microbiol. 3:1833-1841) with modifications (Lee,et al., 1990, PNAS 87:4304-4308). Bioluminescence assays were performedessentially like the CFU assays, except that the number of cells wasquantitated using bioluminescence, as opposed to CFUs.

Briefly, HEp-2 cells and primary murine peritoneal macrophages wereseeded into 24-well tissue culture dishes at 1×10⁵ cells per well inRPMI (Gibco/BRL, Grand Island, N.Y.) supplemented with 20 mM glutamine(Gibco/BRL) and 5% fetal calf serum (Hyclone, Logan, Utah). Twenty fourhours (HEp-2) or seven days (macrophages) after cell seeding, bacteriafrom static cultures (see “Materials and Methods”, above) wereinoculated at 1×10⁶ (multiplicity of infection (m.o.i.) of 10) or 1×10⁷(m.o.i. of 100, columns on right in FIGS. 2B, 2C, 2D, and 2E) organismsper well and centrifuged onto the cell monolayer for 5 minutes at 1000rpm (185×g) in a Beckman clinical centrifuge (Beckman Instruments,Columbia, Md.). The medium was replaced with RPMI medium (Gibco/BRL)either with (entry assay) or without (adherence assay) gentamicin (100mg/ml). The co-cultures were incubated for a total of 3.5 hours at 35°C. in 5% CO₂.

Gentamicin in the incubation medium kills bacteria that had not beeninternalized by the HEp-2 cells, including those adhering to thesurfaces of the HEp-2 cells. Accordingly, the signal in adherence assays(without gentamicin) represent both adherent and internalized bacteria,whereas the signal in entry assays (with gentamicin) represent onlyinternalized bacteria.

Adherence and entry were assayed by imaging luminescent bacterial cellsat three timepoints—1.5, 3.0 and 3.5 hours post inoculation. Prior toimaging at the first timepoint, the cell monolayer was washed threetimes with phosphate-buffered saline (PBS) to remove unattached bacteriaand a fresh aliquot of RPMI medium was added. Luminescence was recordedusing a 30 second exposure. Images at the second and third timepointswere obtained using a similar exposure, but without first washing thecells.

Data recorded at the last timepoint, displayed as pseudocolorluminescence images superimposed over gray scale images of the culturedish wells, are shown in FIG. 2A. The cell types, Salmonella strains,and usage of gentamicin are indicated in the FIGURE. The data are alsosummarized as relative intensity of photon counts in the graphs in FIGS.2B and 2D.

Following imaging at the 3.5 hour timepoint, the tissue culture cellswere washed three times with PBS and lysed with 0.2% “TRITON X-100” inPBS. Adherent and/or intracellular bacteria, released by lysis, wereplated on LB- or LB-carbenicillin agar plates and incubated for 18 h at35° C. The number of bacteria released from each well was determined bycounting the number of colony forming units (CFU, Finlay and Falkow,1989, Mol. Microbiol. 3:1833-1841. Lee, et al., 1990, PNAS87:4304-4308). These data are represented as the total bacterialcolonies per ml recovered from co-culture after incubation for 3.5 hwith or without gentamicin, and are summarized in the graphs in FIGS. 2Cand 2E.

Data from both the bioluminescence and CFU assays indicate that (i)Salmonella transformed with the lux genes have an infective potentialsimilar to that of the parent lines, and (ii) luminescence detection andCFU determination yield comparable estimates for the invasive potentialof the two Salmonella strains in HEp-2 cells and macrophages. The ratioof bioluminescence to CFU was lower in macrophage cultures, possibly dueto the subcellular compartment in which the Salmonella entermacrophages.

D. Example 3 In vitro Luminescence of Transformed Salmonella

10 μl of four 10-fold serial dilutions (ranging from 10⁶ cells to 10³cells per ml) of LB5000lux Salmonella were placed in four 100 μl glasscapillary tubes (Clay-Adams div. of Becton Dickinson, Parsippany, N.J.).The bacterial suspensions formed columns of fluid in the tubes, withpockets of air at both ends. One end of each tube was sealed withcritoseal (Clay-Adams). The medium in which dilutions were made wassaturated with O₂ through exposure to air.

The tubes were wrapped with clear plastic wrap and luminescence wasdetermined by imaging for 30 seconds as described above. An exemplaryimage is shown in FIG. 3A. Four tubes are pictured. They contained (fromtop to bottom) 10⁶, 10⁵, 10⁴ and 10³ Salmonella cells/ml (10⁴, 10³, 10²and 10 cells/tube). Luminescence could be detected in suspensionscontaining as few as 10⁴ cells/ml (100 cells). The luminescence isconfined, however, to air/liquid interfaces, suggesting that theluminescence reaction requires relatively high levels of oxygen. Sincemany of the cells are presumably in the fluid column and not at theair/fluid interfaces, the data suggest that the luminescence in thecapillary tubes shown in FIG. 3A arises from considerably fewer than thetotal number of cells in each tube.

E. Example 4 In vitro Detection of Luminescence through Animal Tissue

Micro test-tubes, constructed from glass capillary tubing with aninternal diameter of 3.5 mm, containing serial dilutions of LB5000luxSalmonella were prepared essentially as described in Example 3, above.In the present example, however, the bacterial suspensions contacted thesealed end of the tube and were exposed to air only at the upper end.The tubes were placed in a translucent plastic scintillation vial andsurrounded by one of the following animal tissues: chicken breastmuscle, chicken skin, lamb kidney or lamb renal medulla. All tissueswere obtained from the meat department of a local supermarket (Safeway,Mountain View, Calif.).

A diagram of a vial containing a capillary tube surrounded by tissue isshown in FIG. 5. The vial 1 is approximately 1.4 cm in diameter andincludes a cap 2. The vial is coated with an opaque material (i.e.,black tape) along its upper portion 3. Animal tissue 4 is placed in thevial such that it extends from the bottom of the vial to just above thebottom edge of the opaque coating 3. The micro test-tube 5 is sealed atthe bottom by a plug 7 (i.e., a crytoseal plug), and is centeredradially in the vial, with the plugged end of the tube touching or inclose proximity to the bottom of the vial. The bacterial suspension 6extends approximately 1 cm upward from the bottom of the tube.

Photons emitted from vials with and without tissue, and with and withoutbacteria, were counted using a liquid scintillation counter (model 1219Rackbeta, LKB/Wallac, Gaithersburg, Md.) with the fast coincidencediscriminator disabled.

Controls without tissue were assayed by placing the bacterial suspensiondirectly in the scintillation vial. All experiments were performed intriplicate.

In each experiment, the vials were counted two to three times, rotatingthe vial 90° between each count, to control for effects of possibletissue thickness inconsistency. No significant differences weredetected.

The results are summarized in TABLE I, below.

TABLE I TRANSMISSION OF PHOTONS THROUGH TISSUE Chicken Chicken Lamb LambSample skin muscle kidney medulla Vial alone 2.1 × 10⁴ 1.3 × 10⁴ 1.0 ×10⁴ 1.0 × 10⁴ Tissue alone N.D. 1.5 × 10⁴ 9.4 × 10³ 8.5 × 10³ Tissue and2.7 × 10⁵ 2.3 × 10⁵ 1.6 × 10⁴ 1.5 × 10⁵ LB5000lux* LB5000lux* 2.0 × 10⁶1.7 × 10⁶ 4.8 × 10⁶ 4.8 × 10⁶ alone Counts are averages of triplicatemeasurements, tissue path length was 1 cm. *1 × 10⁷ cells.

The signal for 1×10³ LB5000lux in kidney tissue was at or nearbackground levels using, the photomultiplier tubes (PMT) in thescintillation counter. The background in this type of detection is dueto the dark current of the PMT and limits the studies to analysis ofrather intense signals.

Bioluminescence from approximately 1×10⁷ LB5000lux was detectablethrough 0.5 cm of avian muscle, skin ovine renal medulla and ovinekidney. These results indicate that bioluminescence from the labeledSalmonella was detectable through animal tissues of variable opacity.Since oxygen was likely limited in the capillary tubes (as demonstratedin FIG. 3A), it is likely that fewer numbers of bioluminescentSalmonella could be detected through tissue than are indicated in thisassay.

F. Example 5 In vivo Detection of Bioluminescent Salmonella

To assess the availability of oxygen to Salmonella during infection,wild-type SL1344lux was inoculated into the peritoneal cavity (i.p.) ofBALB/c mice. Photons emitted from the bacteria internally, andtransmitted through the abdominal wall were externally detected andlocalized in anaesthetized mice using an intensified CCD camera 24 hafter inoculation (FIG. 3B). Systematic Salmonella infections arethought to involve colonization of the lymph nodes, spleen, liver forVentral images of the mice infected by i.p. inoculation of wild-type SL1344lux demonstrated transmitted photons over much of the abdominalsurface, with foci of various intensities (FIG. 3B). These results wereconsistent with widespread colonization of the viscera, possiblyincluding the liver and mesenteric lymph nodes, and indicate that thelevel of available oxygen in some tissues can be adequate for externaldetection of bioluminescence from the labelled pathogen.

G. Example 6 Effect of Human Blood on the Light Emission fromBioluminescent Salmonella

As demonstrated in the following example, fewer than ten (10) bacterialcells can be detected with an intensified CCD detector.

Two fold serial dilutions of Salmonella, strain LB5000, that had beentransformed with a plasmid that conferred constitutive expression of theluciferase operon were plated in duplicate into 96 well plates.Dilutions were made in 30 μl of growth medium alone (indicated asLB5000) and with 30 μl of blood to determine the effects of blood as ascattering and absorbing medium on the limits of detection. Eachdilution and the numbers of colony forming units (CFU) implied fromplating samples from concentrated wells are indicated in FIG. 4. Therelative bioluminescence for each well as determined by analysis of theimage generated by the CCD detector is shown (Table III). The signal inthe more concentrated wells was off scale and the numbers are thereforenot linear at higher concentrations.

TABLE III Relative Bioluminescence Dilution LB5000 LB5000 and 30 μlblood 1:100  199243 159497 1:200  187163 110081 1:400  170044 722341:800  154031 46273 1:1600 146934 17598 1:3200 112196 6731 1:6400  50302320

H. Example 7

Detection of Orally-Administered lux Salmonella in Balb/c Mice

Balb/c mice were infected by oral feeding (Stocker, et al.) with a 50 μlsuspension of 1×10⁷ virulent SL1344lux, non-invasive BJ66lux and lowvirulence LB5000lux Salmonella . The mice, 4-6 weeks of age at the timeof infection, were imaged daily with 5 minute integration times (photonemission was measured for 5 minutes). Prior to imaging, the mice wereanesthetized with 33 μg/kg body weight nembutal.

Representative images are shown in FIGS. 6A, 6B, and 6C. At 24 hourspost inoculation (p.i.), the bioluminescent signal localized to a singlefocus in all infected animals (FIGS. 6A, 6B, and 6C). Bioluminescencedisappeared in all animals infected with the low virulence LB5000lux by7 days p.i. (FIG. 6A). In BALB/c mice infected with the wild-typeSL1344lux, bioluminescence was detected throughout the study period,with multiple foci of transmitted photons at 8 d. In these animals, theinfection frequently spread over much of the abdominal cavity (FIG. 6C).In one-third of these animals, transmitted photons were apparent overmuch of the abdominal area at 8 d, resembling the distribution ofphotons following an i.p. inoculation (see FIGS. 3B and 6F). The spreadof infection by BJ66lux was more variable, but the infection typicallypersisted and remained localized at the initial site (FIG. 6B).

After infection of resistant BALB/c×129 mice with wild-type SL 1344lux,the bioluminescent signal remained localized and persistent in a groupof 10 mice throughout the study period. This result was in contrast tothe disseminated bioluminescence observed in SL1344lux-infectedsusceptible mice (lty^(r/s)) (see, Example 9 and FIGS. 8A and 8B), butresembled the persistent infection of susceptible BALB/c mice with theless invasive BJ66lux. As a control, Salmonella were cultured frompersistently infected resistant BALB/c×129 mice, and 80-90% of thecolonies recovered after 8 d were Amp^(r). Of these, more than 90% werebioluminescent, suggesting that observed differences were not due tosignificant loss of lux plasmid, but rather were due to real differencesin pathogenicity of the bacterial strains.

I. Example 8 Detection of Infection Following I.P. Inoculation with aVirulent and a Low Virulence Strain of Salmonella

Balb/c mice were infected with either virulent (SL1344lux) or lowvirulence (LB5000lux) Salmonella by intraperitoneal (i.p.) inoculationsof 1×10⁷ bacterial cells in a 100 μl suspension, without simultaneousinjection of air.

At 32 hours post injection (p.i.), the mice were anesthetized and imagedas described above. The results are shown in FIG. 7. Widespreadinfection is evident in the two mice in the left part of FIG. 7,infected with the virulent SL1344lux strain. In contrast, little, ifany, luminescence is detected in the mice on the right, injected withthe low virulence LB5000lux strain.

J. Example 9 Detection of Systemic Infection in Resistant Mice FollowingOral Inoculation with Salmonella

Resistant 129×Balb/c (Ity^(r/s)) viable mice were infected byintragastric inoculation of 1×10⁷ SL1344lux Salmonella. The bacteriawere introduced through an intra-gastric feeding tube while underanesthesia. The animals were imaged daily for 8 days post injection(d.p.i.).

Results are shown in FIGS. 8A and 8B. Mice, in triplicate, were infectedand imaged daily for 8 days. Exemplary images for day 1 (FIG. 8A) andday 8 (FIG. 8B) are shown. These data indicate that mice resistant tosystemic Salmonella infection have a localized chronic infection in thececum, but that the infection does not spread into the abdominal cavity.

K. Example 10 Post-Laparotomy Imaging Following Oral Inoculation withSalmonella

Laparotomy was performed following oral inoculation of Salmonella toprecisely localize the luminescent signal within the abdominal cavity,and to compare this localization with that obtained using non-invasiveimaging. The animals were inoculated as described in Example 9. After aselected period of time, typically seven days, the mice wereanesthetized and externally-imaged, as described above. An exemplaryimage is shown in FIG. 9A. After external imaging, the peritoneal cavitywas opened and the animals were imaged again, as illustrated in FIG. 9B.In some instances the mice were imaged a third time, following injectionof air into the lumen of the intestine both anterior and posterior tothe cecum (C) (FIG. 9C). The mice were euthanized immediately after thefinal imaging.

In each case where a focal pattern of bioluminescence was observed insusceptible mice, early in infection after oral inoculation, photonsoriginated almost exclusively from the cecum, while variations in theprecise localization and intensity of focal bioluminescence were due tovariable positioning of the cecum. The focal pattern of bioluminescenceobserved in infection-resistant BALB/c×129 mice similarly localized tothe cecum. In contrast, such localization was not observed in animalsinfected i.p. with SL1344lux (FIG. 3B). At late stages ininfection-susceptible mice inoculated orally with the wild-typeSL1344lux, bioluminescence was multifocal, however, additional foci ofluminescence did not become apparent after laparotomy. In mice infectedwith the less-virulent LB5000lux, bioluminescence was not detectable at7 d in any tissue or organ, even focally, after removal of the skin andperitoneal wall.

Bioluminnescence was not detected optically in the spleen or bloodstreamof any infected animal; bioluminescence from the liver was seen only atlater stages of disease; and bioluminescence from the G.I. tract wasrestricted to the cecum early in the disease course. This pattern couldbe due to differences in the numbers of Salmonella in the differenttissues, or lack of available oxygen. The Amp^(r) cfu present inhomogenized organs of orally infected mice were quantified to evaluatethe distribution of labelled Salmonella SL1344lux. Greater than 90% ofthe amp^(r) bacterial colonies obtained from all analyzed tissues ofSL1344lux-infected BALB/c mice at 7 d indicated total cfu from theliver, spleen, and lungs were in the range of 1.9×10³ to >1.0×10⁵without detectable photon emission, in vivo (TABLE II). In contrast,bioluminescence was detectable from the cecum and this tissue contained>1.0×10⁸ total cfu. No cfu were detectable in any tissue of theLB5000lux infected mice. These results suggest that 1×10⁶ organisms intissue is near the limit of detection at this emission wavelength usingthe current experimental system.

Oxygen is an essential substrate for the luciferase reaction, thus onlySalmonella present in oxygenated microenvironments should bebioluminescent. The absence of bioluminescence from Salmonella in theanaerobic environment of the lumen of the G.I. tract is thereforepredictable, and exposure of the intestinal lumen to air should revealthe presence of bacteria previously not detectable due to a lack ofoxygen. In support of this view, one animal with detectablebioluminescence in the cecum alone excreted a faecal pellet that rapidlybecame bioluminescent upon exposure to air. This indication ofnon-luminescent, luciferase-expressing bacteria in the lumen of theintestine and the clear delineation of the aerobic and anaerobic zonesin this tissue, suggested that injection of air into the lumen of theintestine would reveal the presence of additional bacteria. Injection ofair into the lumen of the ileum and colon of another animal, with asimilar pattern of bioluminescence, resulted in detectable photons nearthe injection sites (FIG. 9). Last, when a third mouse with cecalbioluminescence was killed, bioluminescence quickly ceased. Air wasinjected at other tissue sites because of the lack of clear zones ofaerobic and anaerobic environments.

TABLE II Colony-forming units in homogenized tissue from mice infectedwith bioluminescent Salmonella Tissue Animal Weight Total Strain TissueNumber (mg) cfu SL1344lux Liver 1 441 1.9 × 10³ 2 778 2.5 × 10⁴ Spleen 1218 1.2 × 10⁴ 2 248 4.9 × 10⁵ Mesenteric 1 76 >1.0 × 10⁶  lymph node 246 >1.0 × 10⁶  Lung 1 17 1.5 × 10³ 2 69 2.7 × 10³ Cecum 1 351 >1.0 ×10⁸* 2 422 >1.0 × 10⁸* *Photons emitted from bacteria at these tissuesites were externally detected.

L. Example 11 Post-Laparotomy Imaging Following I.P. Inoculation withSalmonella

Balb/c mice were infected by intraperitoneal inoculation of 1×10⁷Salmonella (SL1344lux) as described in Example 8. Exemplary images ofone such animal are shown in FIGS. 10A, 10B and 10C.

At 24 hours post-injection (p.i.), the animal was anesthetized andimaged for five minutes (FIG. 10A). The peritoneal cavity was opened andthe mouse was imaged again for five minutes (FIG. 10B). The cecum waspulled to the left side, and the animal was again imaged for fiveminutes (FIG. 10A).

The results demonstrate that the localization of infection sitesobtained with non-invasive imaging correlates well with the sites asrevealed upon opening the peritoneal cavity.

M. Example 12 Effects of Ciprofloxacin Treatment on Bioluminescence fromSL1344lux Salmonella

To demonstrate the utility of in vivo imaging, an infected animal wastreated with the antibiotic ciprofloxacin, which known to be effectiveagainst systemic Salmonella infections. Magalianes, et al., 1993,Antimicrobial Agents Chemo. 37:2293.

Experimental and control groups of Balb/c mice were orally inoculatedwith SL1344lux. At 8 days p.i., mice in the experimental group wereinjected i.p. with 100 mg of ciprofloxacin hydrochloride (3 mg/kg bodyweight; Sigma Chemical Co., St. Louis, Mo.). Following treatment of theexperimental group, animals from both groups were imaged (as above) atseveral intervals over a period of 5.5 h post treatment.

Representative images are shown in FIGS. 11B, 11C, 11D, and 11E. FIGS.11B and 11D show composite images of representative animals from thecontrol and treated groups, respectively, immediately before initiationof treatment of the experimental group. FIGS. 11C and 11E show compositeimages of the same animals 5.5 hours after initiation of treatment.Bioluminescence over the abdomen of the ciprofloxacin-treated animal wasreduced to undetectable levels during this period of time, whilebioluminescence in the control typically increased 7.5-fold. The totalnumber of photons detected over the abdominal area were determined,normalized to the value at t=0, and plotted in FIG. 11A with respect totime post-treatment.

The data demonstrate that methods and compositions of the presentinvention can be used to evaluate the effects of drugs on the spread ofinfection in vivo.

N. Example 13 Bioluminescent Reporter for Promoter Activity in CulturedCells

In order to demonstrate how the promoter from HIV (humanimmunodeficiency virus) responds to viral infection over time, jurketcells transfected with a plasmid containing the HIV LTR (long terminalrepeat, promoter) upstream of the coding sequence of firefly luciferasewere infected with a laboratory isolate of HIV-1 (strain A111) usingstandard laboratory conditions and followed for a period of 7 days (d)for emission of bioluminescent light. After 24 h, 60 h, 96 h, and 7 d, agray scale image of the plate was generated in low room light followedby collection of photons emitted from the cultured cells in completedarkness for a period of 10 min. A color pseudoimage representing theintensity of bioluminescent light was superimposed over the gray scaleimage of the plate (FIG. 12). At 7 d post infection a clear signal ispresent in the duplicate cultures indicating high levels of replication(FIG. 12). The images at different time points represent the same twowells. Advantages of this assay for HIV replication are that: i)temporal studies can be done in a minimum number of wells since the samewells are followed over time, ii) kinetics of replication can thereforebe studied as a phenotypic characteristic of viral isolates, iii)samples of cells or supernatant do not have to be collected, iv) thelevel of viral replication is almost immediately apparent, v) thedetection could be set up remotely limiting human handling, vi)antiviral drugs could be evaluated in culture with the above listedadvantages.

O. Example 14 Assessing Promoter Activity in Tissues of Transgenic Mice

Transgenic mice containing a construct composed of the regulatoryportion of the HIV LTR (U3 region) upstream of the coding sequence ofthe firefly luciferase gene were generated and evaluated for theemission of photons after transmission through tissues. A diagram of theconstruct is shown at the bottom of FIG. 13. Numbers along the constructindicate nucleotide positions relative to the start of transcription.Sequences matching known motifs of cellular transcription factors areindicated with the names of the factors. Transcription from the HIV LTRwas activated in the right ear of each two animals with a single topicalapplication of dimethyl sulfoxide (DMSO). The animal on the left in FIG.13 was given 150 μl of an aqueous solution of the substrate luciferin(50 mg/ml) via intraperitoneal (i.p.) injection. The animal on the rightwas not given substrate. 20 min. post treatment with substrate theanimals were imaged as described for the plate in FIG. 12 with a 20 min.integration time. The color pseudoimage indicates light emission overthe right ear (B) of the animal on the left, and not from the uninducedear (A) or the animal that was not given substrate (C,D). This is thefirst demonstration of monitoring promoter activity in a living adultanimal and demonstrates the relatively tight regulation of the LTR withDMSO induction. This technology allows for the temporal and spatialanalyses of transcriptional activity in living animals.

P. Example 15 Topical Delivery of Substrate to Dermal Cells inTransgenic Animals

In order to optimize delivery of substrate, the substrate was topicallydelivered to dermal cells. The HIV LTR was induced in the skin of micewith twice daily treatments of DMSO over the entire surface of the backand the right ear for two consecutive days. Substrate was applied to theskin in solutions prepared in DMSO. Concentrations included 200 mM, 100mM, 50 mM, 25 mM, and 12.5 mM. 5 μl of each concentration were spotted,in quadruplicate, on the backs using a multichannel pipette with thehighest concentrations near the head. 5 μl of the 50 mm solution wasapplied to each ear. 2 min. after application the animal was imaged asdescribed in FIG. 12. The bioluminescent response appeared increaselinearly over the concentrations from 12.5 mM to 100 mM (FIG. 14).Bioluminescence from spots containing 200 mM luciferin was roughlyequivalent to that from the 100 mM spots, solutions of luciferincontaining water, in contrast, resulted in no detectable bioluminescence(FIG. 14). Solutions of 25% H₂O in DMSO to 100% H₂O were tested.

Q. Example 16 Induction of Bioluminescence in Ears of Transgenic Animalsby Topical Luciferin Delivery

The experiment of induction of luciferase expression in ears andsystemic luciferin delivery of Example 14 was repeated with topicaladministration of substrate in 100% DMSO (FIG. 15). Signals from theears were uniform and had greater intensity than with systemic luciferindelivery. See, FIGS. 13 and 15. Peak light emission was observedimmediately after topical treatment compared to 20-30 min. aftersystemic administration of substrate.

R. Example 17 Unilateral Induction of Luciferase Expression inTransgenic Mice

The left half of the shaved dorsal surface of the transgenic animals andthe left ear were treated twice daily for two days with DMSO to activateexpression of the HIV-1 LTR. Luciferin was applied topically over theentire surface of the back and both ears, and animals were imagedimmediately after addition of substrate. Unilateral emission ofbioluminescence corresponding to the induced region was observed (FIG.16).

S. Example 18 Bioluminescence Detectable in Internal Tissues ofTransgenic Animals

Bioluminescence was detectable from the abdomens of animals treated withDMSO on one ear only. This signal is assumed to be due to ingestion ofDMSO during grooming (FIG. 17).

T. Example 19 Localization of Internal Bioluminescence in TransgenicMice

Animals demonstrating signal from the abdomen were laprotomized andimaged. Bioluminescent signal localized to the colon in 4 of 4 animalsstudied and in the animals shown in FIG. 18 was tightly localized to aregion of the colon about 1 cm in length. In the other animals theentire colon appeared to emit bioluminescent light.

U. Example 20 Expression of the HIV-LTR in Neonatal Transgenic Mice

As the demonstrated in the following experiment, the HIV-LTR isdifferentially expressed through development.

4 d old transgenic mice were given intraperitoneal injections ofluciferin in aqueous solution (15 μl at 50 mM), and imaged withintegration times of 20 min. In the absence of any known inducing agentor treatment, bioluminescent signal indicative of expression ofluciferase form the HIV LTR was apparent as a diffuse signal over muchof the surface of the animal with more intense signal originating fromthe developing eye and extremities (FIG. 19). These data demonstratethat the LTR is inherently active in neonatal transgenic mice, and maybe expressed to a greater level in the eyes and other locations.

All references are hereby incorporated in their entirety.

1. A non-invasive method for detecting expression of a heterologous genein a living, non-human, mammalian subject, said method comprisingproviding a mammalian subject whose cells comprise a transgene, wherein(i) said transgene comprises a heterologous gene that encodes afluorescent protein, (ii) expression of the heterologous gene ismediated by a promoter and (iii) said subject comprises opaque tissue;and measuring photon emission through opaque tissue of said subjectwherein said photon emission is mediated by excitation of saidfluorescent protein expressed from said heterologous gene.
 2. The methodof claim 1, wherein said measuring is done using a photodetector device.3. The method of claim 2, wherein said measuring is carried out with anintensified charge-coupled photodetector device.
 4. The method of claim2, wherein said measuring is carried out with a cooled charge-coupledphotodetector device.
 5. The method of claim 1, wherein said measuringis carried out using fiber optic cables.
 6. The method of claim 5,wherein said fiber optic cables terminate in a tightly-packed array. 7.The method of claim 5, wherein said fiber optic cables detect light froma limited defined region of the subject.
 8. The method of claim 1,wherein said fluorescent protein is green fluorescent protein, lumazine,or yellow fluorescent protein.
 9. The method of claim 1, wherein aninput of light to excite the fluorescent protein is produced by a laser.10. The method of claim 1, further comprising constructing a photonemission image from said measured photon emission.
 11. The method ofclaim 10, further comprising acquiring a reflected light image of thesubject, and superimposing said image of photon emission on saidreflected light image to form a composite image.
 12. The method of claim1, wherein said expression of the heterologous gene is mediated by aconstitutively active promoter.
 13. The method of claim 1, wherein saidexpression of the heterologous gene is mediated by an induciblepromoter.
 14. The method of claim 13, wherein said inducible promoter isan interferon-inducible promoter.
 15. The method of claim 13, whereinsaid inducible promoter is a promoter expressed in a disease state or atissue-specific promoter.
 16. The method of claim 15, wherein saiddisease state is inflammation.
 17. The method of claim 16, wherein saidpromoter is E-selectin.
 18. The method of claim 15, wherein said diseasestate is a tumor.
 19. The method of claim 1, wherein expression of saidheterologous gene is by a tumor cell.
 20. The method of claim 1, furthercomprising: repeating said detecting at selected intervals, wherein saidrepeating is effective to track localization of the expression of theheterologous gene in the subject over time.
 21. The method of claim 1,further comprising administering a compound to said subject, andmeasuring photon emission from said subject after administration of saidcompound.
 22. The method of claim 21, further comprising: repeating atselected intervals said measuring after administration of said compound,wherein said repeating is effective to track an effect of said compoundon a level of expression of said heterologous gene in said subject overtime.
 23. The method of claim 1, wherein said method fluiher comprises,prior to said detecting, placing the subject in a detection field of thephotodetector device.
 24. The method of claim 1, wherein photons thatmake up said photon emission are visible light photons.
 25. The methodof claim 1, wherein said measuring consists of measuring photon emissionfrom within the subject with a photodetector device located outside ofthe subject.
 26. The method of claim 25, wherein the measuring is doneusing a photodetector device.
 27. The method of claim 26, wherein saidmeasuring is canied out with an intensified charge-coupled photodetectordevice.
 28. The method of claim 26, wherein said measuring is carriedout with a cooled charge-coupled photodetector device.
 29. The method ofclaim 25, wherein said measuring is carried out using fiber opticcables.
 30. The method of claim 29, wherein said fiber optic cablesterminate in a tightly-packed array.
 31. The method of claim 29, whereinsaid fiber optic cables detect light from a limited defined region ofthe subject.
 32. The method of claim 25, wherein said fluorescentprotein is green fluorescent protein, lumazine, or yellow fluorescentprotein.
 33. The method of claim 25, wherein an input of light to excitethe fluorescent protein is produced by a laser.
 34. The method of claim25, further comprising constructing a photon emission image from saidmeasured photon emission.
 35. The method of claim 10, further comprisingacquiring a reflected light image of the subject, and superimposing saidimage of photon emission on said reflected light image to form acomposite image.
 36. The method of claim 25, wherein said expression ofthe heterologous gene is mediated by a constitutively active promoter.37. The method of claim 25, wherein said expression of the heterologousgene is mediated by an inducible promoter.
 38. The method of claim 37,wherein said inducible promoter is an interferon-inducible promoter. 39.The method of claim 37, wherein said inducible promoter is a promoterexpressed in a disease state or a tissue-specific promoter.
 40. Themethod of claim 39, wherein said disease state is inflammation.
 41. Themethod of claim 40, wherein said promoter is E-selectin.
 42. The methodof claim 39, wherein said disease state is a tumor.
 43. The method ofclaim 25, wherein expression of said heterologous gene is by a tumorcell.
 44. The method of claim 25, further comprising: repeating saiddetecting at selected intervals, wherein said repeating is effective totrack localization of the expression of the heterologous gene in thesubject over time.
 45. The method of claim 25, further comprisingadministering a compound to said subject, and measuring photon emissionfrom said subject after administration of said compound.
 46. The methodof claim 45, further comprising: repeating at selected intervals saidmeasuring after administration of said compound, wherein said repeatingis effective to track an effect of said compound on a level ofexpression of said heterologous gene m said subject over time.
 47. Themethod of claim 25, wherein said method further comprises, prior to saiddetecting, placing the subject in a detection field of the photodetectordevice.
 48. The method of claim 25, wherein photons that make up saidphoton emission are visible light photons.